参见作者原研究论文

本实验方案简略版
Dec 2021

本文章节


 

A Highly Sensitive Method to Efficiently Profile the Histone Modifications of FFPE Samples
一种高效分析 FFPE 样品组蛋白修饰的高灵敏度方法   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

The majority of biopsies in both basic research and translational cancer studies are preserved in the format of archived formalin-fixed paraffin-embedded (FFPE) samples. Profiling histone modifications in archived FFPE tissues is critically important to understand gene regulation in human disease. The required input for current genome-wide histone modification profiling studies from FFPE samples is either 10–20 tissue sections or whole tissue blocks, which prevents better resolved analyses. Nevertheless, it is desirable to consume a minimal amount of FFPE tissue sections in the analysis as clinical tissue of interest are limited. Here, we present FFPE tissue with antibody-guided chromatin tagmentation with sequencing (FACT-seq), highly sensitive method to efficiently profile histone modifications in FFPE tissue by combining a novel fusion protein of hyperactive Tn5 transposase and protein A (T7-pA-Tn5) transposition and T7 in vitro transcription. FACT-seq generates high-quality chromatin profiles from different histone modifications with low number of FFPE nuclei. We showed a very small piece of FFPE tissue section containing ~4000 nuclei is sufficient to decode H3K27ac modifications with FACT-seq. In archived FFPE human colorectal and human glioblastoma cancer tissue, H3K27ac FACT-seq revealed disease specific super enhancers. In summary, FACT-seq allows researchers to decode histone modifications like H3K27ac and H3K27me3 in archival FFPE tissues with high sensitivity, thus allowing us to understand epigenetic regulation.


Graphical abstract:



(i) FFPE tissue section; (ii) Isolated nuclei; (iii) Primary antibody, secondary antibody and T7-pA-Tn5 bind to targets; (iv) DNA purification; (v) In vitro transcription and sequencing library preparation; (vi) Sequencing


Keywords: FFPE tissue (FFPE组织), Histone modifications (组蛋白修饰), High sensitivity (高灵敏度), T7-pA-Tn5 transposase (T7-pA-Tn5 转座酶), FACT-seq (FACT-seq)

Background

Currently, Formalin-Fixed Paraffin-Embedding (FFPE) is a universal approach for biopsy specimen processing in basic research and translational studies (Fox et al., 1985; Wang et al., 2005 ; Haile et al., 2019). Furthermore, it has been reported that large numbers of FFPE specimens are archived worldwide each year (Waldron et al., 2012). More and more techniques have been developed to decipher genomic(Astolfi et al., 2015; Bolognesi et al., 2016), transcriptomic (Pennock et al., 2019 ), and proteomic (Becker et al., 2017) information in FFPE samples. Nevertheless, a method that can profile epigenetic regulation in FFPE samples with high sensitivity is lacking. Previously, researchers applied chromatin immunoprecipitation with sequencing (ChIP-seq) to archived FFPE tissue. And they have successfully developed pathology tissue chromatin immunoprecipitation (PAT-ChIP) (Fanelli et al., 2010, 2011), fixed-tissue chromatin immunoprecipitation sequencing (FiT-seq) (Cejas et al., 2016), and fixed-tissue ChIP-seq for H3K27 acetylation profiling (FiTAc-seq) (Font-Tello et al., 2020) making it possible to profile histone modifications in FFPE tissue. However, the required input for these technologies from FFPE samples is either 10–20 tissue sections or whole tissue blocks. A higher sample consumption prevents them from being used in a broader application since clinical samples are usually limited. In addition, sonication is included in these available methods, which may potentially introduce sequencing bias (Teytelman et al., 2009). Here, we present a new highly sensitive method, we recently developed, to efficiently profile the histone modifications from FFPE samples using antibody-guided chromatin tagmentation with sequencing (FACT-seq) (Zhao et al., 2021). FACT-seq uses a novel fusion protein (T7-Pa-Tn5) that combines hyperactive Tn5 transposase with protein A, utilizing T7 in vitro transcription to prepare a library, and profile histone modifications in a small amount of FFPE sample.

Materials and Reagents

  1. 50 mL Falcon tubes (Sarstedt, catalog number: 62.559.001)

  2. 21 G needles (BD Microlance, catalog number: ND432)

  3. 27 G needles (BD Microlance, catalog number: 302200)

  4. 1 mL syringes (BD Microlance, catalog number: 309628)

  5. 1.5 mL Low-bind tubes (Sarstedt, catalog number: 72.706.600)

  6. 0.5 mL Qubit tubes (Invitrogen, catalog number: Q32856)

  7. 30 μm Filters (Miltenyi Biotech MACS, catalog number: 130-098-458) (room temperature)

  8. Xylene (Histolab, catalog number: 2070) (Room temperature)

  9. Ethanol (VWR BDH Chemicals, catalog number: VWRC20816.552) (room temperature)

  10. Collagenase (Sigma-Aldrich, catalog number: C9263)(-20°C)

  11. Hyaluronidase (Merck Millipore, catalog number: HX0154) (-20°C)

  12. Ampicillin (Serva, catalog number: 69-52-3) (+4°C)

  13. Sodium azide (Merck Millipore, catalog number: 822335) (room temperature)

  14. BSA (Miltenyi Biotech MACS, catalog number: 130-091-376) (+4°C)

  15. IGEPAL CA-630 (Sigma-Aldrich, catalog number: I3021) (+4°C)

  16. 1 M CaCl2 (Alfa Aesar, catalog number: J63122) (room temperature)

  17. 5 M NaCl (Thermo Fisher Scientific, Invitrogen, catalog number: AM9759) (room temperature)

  18. 1 M Tris-HCl pH 8.0 (Thermo Fisher Scientific, Invitrogen, catalog number: 15568-025)

  19. 1 M Tris-HCl pH 7.5 (Thermo Fisher Scientific, Invitrogen, catalog number: 15567-027)

  20. 1 M MgCl2 (Thermo Fisher Scientific, Invitrogen, catalog number: AM9530G) (room temperature)

  21. FBS (Life Technologies, catalog number: 10108-105) (-20°C)

  22. RNase A (Thermo Fisher Scientific, catalog number: EN0531) (-20°C)

  23. DTT (Thermo Fisher Scientific, catalog number: 20291) (+4°C)

  24. 0.5 M EDTA (Thermo Fisher Scientific, Invitrogen, catalog number: AM9260G) (room temperature)

  25. Dimethylformamide (Sigma-Aldrich, catalog number: D4551) (room temperature)

  26. Glycerol (Sigma-Aldrich, catalog number: G9012) (Room temperature)

  27. Sodium deoxycholate (Sigma-Aldrich, catalog number: D6750) (room temperature)

  28. Triton X-100 (Sigma-Aldrich, catalog number: T8787) (room temperature)

  29. Human genomic DNA (Promega, catalog number: G3041) (+4°C)

  30. Qiagen miniElute PCR purification kit (Qiagen, catalog number: 28004)

  31. 6× Loading Dye (Thermo Fisher Scientific, catalog number: R0611) (-20°C)

  32. HEPES (Sigma-Aldrich, catalog number: H3375)

  33. Protease inhibitor (Sigma-Aldrich, catalog number: 11873580001)

  34. Spermidine (Sigma-Aldrich, catalog number: S2626)

  35. 10% SDS (Thermo Fisher Scientific, Invitrogen, catalog number:1553-035)

  36. Anti-H3K27ac antibody (Abcam, catalog number: ab4729)

  37. Anti-H3K27me3 antibody (Cell Signalling Technology, catalog number: 9733S)

  38. Anti-H3K4me1 antibody (Abcam, catalog number: ab176877)

  39. Anti-H3K36me3 antibody (Abcam, catalog number: ab9050)

  40. Mouse IgG1, kappa monoclonal (Abcam, catalog number: ab18443)

  41. Guinea Pig anti-Rabbit IgG antibody (Antibodies-Online, catalog number: ABIN101961)

  42. Proteinase K (Thermo Fisher Scientific, catalog number: EO0491)

  43. Phenol (Thermo Fisher Scientific, catalog number: 17914)

  44. Chloroform (Sigma-Aldrich, catalog number: C2432)

  45. NEBNext high-fidelity 2× PCR master mix (New England Biolabs, catalog number: M0541S) (-20°C)

  46. SPRIselect beads (Beckman Coulter, catalog number: B23317) (Room temperature)

  47. T7 high yield RNA synthesis kit (New England Biolabs, catalog number: E2040S) (-20°C)

  48. Zymo RNA purification kit (Zymo Research, catalog number: R1013) (room temperature)

  49. SMART MMLV kit (TAKARA, catalog number: 639524) (-20°C)

  50. RNAClean XP beads (Beckman Coulter, catalog number: A63987) (+4°C)

  51. Zymo ChIP DNA clean and concentrator kit (Zymo Research, catalog number: D5205) (room temperature)

  52. 40% Acrylamide:bis-acrylamide (Invitrogen, catalog number: HC2040)

  53. 10% Ammonium persulfate (Invitrogen, catalog number: HC2005)

  54. TEMED (Invitrogen, catalog number: HC2006)

  55. Digitonin (Millipore, catalog number: 300410)

  56. Nuclease-free water (Invitrogen, catalog number: AM9932)

  57. KOH (Sigma-Aldrich, catalog number: 484016)

  58. 50 bp DNA ladder (ThermoFisher Scientific, catalog number: 10488099)

  59. Agilent high sensitive DNA kit (Agilent, catalog number: 5067-4626)

  60. Tn5 transposase [produced in local protein facility following previously description (Picelli et al., 2014)]

  61. pA-Tn5 transposase [produced in local protein facility following previously description (Picelli et al., 2019)]

  62. 1× PBS (pH = 7.4) (Thermo Fisher Scientific, catalog number: 10010023)

  63. SYBR gold (ThermoFisher Scientific, catalog number: S33102)

  64. 10× TBE Buffer (ThermoFisher Scientific, catalog number: B52)

  65. Mouse kidney/liver FFPE tissue blocks (prepared in the lab)

  66. Buffer used in isolation of single nuclei suspension from FFPE tissue (see Recipes)

  67. Buffers used in Tn5 assembly and activity assay (see Recipes)

  68. Buffers used in epitope retrieval (see Recipes)

  69. Buffers used in antibody binding and tagmentation (see Recipes)

  70. Buffer used in library preparation (see Recipes)

Equipment

  1. Microtome (Leica, Histo Core MULTICUT semi-automated rotary microtome, catalog number:149MULTI0C1, 14051856372 or similar one)

  2. Stereo Microscope (Zeiss stemi DV4 stereo microscope 8x-32x, catalog number: 435421-0000-000 or similar one)

  3. ThermoMixer (Eppendorf, Thermo mixer F1.5, catalog number: 5385000016 or similar one)

  4. Thermal Cycler (Applied Biosystems, Veriti, catalog number: 4375786 or similar thermal cycler)

  5. NanoDrop (Thermo Fisher scientific, Nanodrop 2000c, catalog number: ND2000CLAPTOP or similar one)

  6. Cell Counter (Life Technologies, Countess II, catalog number: AMQAF1000 or similar one)

  7. Centrifuge (Eppendorf centrifuge 5415R, catalog number: EP5415R or similar one)

  8. Rotator (Grant InstrumentsTM 360° Vertical Multi-function Rotator PTR 35, catalog number: 9.721028 or similar one)

  9. Microscopy (Zeiss Imager.Z2, catalog number: 490016-0001-000 or similar one)

  10. DynaMagTM-2 magnetic rack (Thermo Fisher Scientific, catalog number: 12321D or similar one)

  11. Agilent DNA Bioanalyzer (Agilent, catalog number: 5067-4626)

  12. Polyacrylamide gel tray (Thermo Fisher Scientific, catalog number: HC1000S)

  13. Gel documentation system (BIO-RAD, catalog number: 1708195EDU)

Software

  1. Bowtie2 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtmL)

  2. Samtools (http://samtools.sourceforge.net/)

  3. Deeptools (https://deeptools.readthedocs.io/en/develop/content/installation.htmL)

  4. SICER (https://zanglab.github.io/SICER2/)

  5. Picard tools (https://broadinstitute.github.io/picard/)

  6. IGV (https://software.broadinstitute.org/software/igv/)

  7. Fiji (https://imagej.net/software/fiji/)

Procedure

  1. Isolation of single nuclei suspension from FFPE block

    1. Sectioning FFPE block into the sections or curls

    2. Deparaffinization and rehydration

    3. Total tissue fragmentation

    4. Disaggregation

    5. Filtration

  2. Tn5 assembly

    1. Tn5 assembly

    2. T7-pA-Tn5 assembly

    3. Tn5 and T7-pA-Tn5 activity assay

  3. Antibody binding and tagmentation

    1. Epitope retrieval

    2. Primary antibody binding

    3. Secondary antibody binding

    4. Tagmentation and reverse cross-linking

  4. DNA isolation and in vitro transcription

    1. DNA purification

    2. Gap filling

    3. SPRI bead purification

    4. In vitro transcription and RNA purification

  5. Reverse transcription and pre-PCR

    1. Reverse transcription

    2. cDNA purification using RNAClean XP beads

    3. Pre-PCR

  6. Second tagmentation and library preparation

    1. Second tagmentation

    2. PCR amplification

    3. Size selection and gel purification

  7. Precise quantification and sequencing

    1. Precise quantification using bioanalyzer

    2. Sequencing


  1. Isolation of single nuclei suspension from FFPE tissue block

    FFPE tissues are processed through various steps: deparaffinization, rehydration, total tissue fragmentation, enzymatic digestion, disaggregation, and filtration to isolate single nuclei suspension.

    1. Sectioning the FFPE block into sections (or curls or slides) and preparing the enzymes.

      Section the FFPE block at various thickness: 7–20 μm.

      Tip: To get good sections without any tissue break, add some ethanol on the top of the tissue block by rubbing with damp ethanol sterile cloth before sectioning it.

    2. Deparaffinization and rehydration

      1. Take one tissue section in a 50 mL Falcon tube, add 1 mL of xylene for deparaffinization, and incubate it for 5 min. After incubation, aspirate the xylene and repeat the same step twice.

      2. After deparaffinization, add 1 mL of 100% ethanol and incubate it for 5 min, followed by sequential rehydration by adding and aspirating different percentages of ethanol (1 mL) (95%, 70%, 50% and 30%). Finally, add 1 mL of nuclease-free water for rehydration and incubate it for 5 min.

    3. Tissue microdissection and enzymatic digestion

      1. After rehydration, the tissue is resuspended in 1 mL of PBS (room temperature) with 0.5 mM of CaCl2, select sections, and use a forceps to transfer tissue to an autoclave glass dish with 200 µL of PBS with 0.5 mM of CaCl2. Perform total tissue fragmentation with two 21 G needles under a stereomicroscope.

        Tip: To get suitable nuclei, make tissue as small as possible.

      2. Transfer the fragmented tissue into a 1.5 mL tube with a 1 mL pipette and centrifuge the sample at 3,000 × g (room temperature) for 10 min. After centrifugation, aspirate the supernatant and add 500 µL of 6 mg/mL collagenase (diluted in PBS with 0.5 mM CaCl2), 500 µL of 600 unit/mL hyaluronidase (diluted in PBS with 0.5 mM CaCl2), 50 μg of sodium azide (Merck Millipore, 26628-22-8), 100 μg of ampicillin and incubate for 16 h at 37°C.

    4. Disaggregation

      1. Prepare fresh NST buffer (Nonidet P40 with Salts and Tris buffer) before starting the experiment (see Table 8 for NST buffer recipe). After 16 h of incubation, add 400 μL of NST buffer and centrifuge the mixture at 3,000 × g for 10 min (room temperature), then aspirate and discard the supernatant.

      2. Add 800 μL of NST buffer,10% fetal bovine serum (88 μL) (on ice), and 0.1% DNase-free RNase A (0.88 μL) (on ice) to the pellet and pass through 2 7G needle syringe for at least 30 times for disaggregation.

    5. Filtration

      1. After disaggregation, centrifuge the mixture at 3,000 × g for 10 min, aspirate supernatant, add 800 μL of NST buffer, and filter the mixture twice using a 30 µm filter.

      2. The filtered mixture should be centrifuged at 3,000 × g for 10 min. Then, discard the supernatant first and resuspend the pellet in 500 μL of 1× PBS, take 5 μL of nuclei suspension to count the cells using a cell counter, take 20 μL of nuclei suspension to stain the nuclei with DAPI, and visualize the single nuclei population (Figure 1).

        Note: 500,000–1,000 000 nuclei could be gotten from one 20 μm-thick mouse kidney section.



      Figure 1. Isolated FFPE mouse nuclei.

      Representative nuclei isolated from 20-µm-thick mouse FFPE kidney tissue sections and stained with DAPI (Scale bar = 10 µm).


  2. Tn5 assembly

    Tn5 assembly mainly covers the steps of loading Tn5 adapter oligos onto Tn5 or pA-Tn5 transposases and checking the Tn5 and T7-pA-Tn5 activity (see Table 1).

    Table 1. Summary of assembled Tn5

    Name Adapters loaded
    Tn5 Tn5ME-A/Tn5MErev + Tn5ME-B/Tn5MErev
    T7-pA-Tn5 T7-Tn5ME/Tn5MErev

    Tip: Prepare 2× dialysis buffer, Tn5, and T7-pA-Tn5 before starting the experiments.

    1. Tn5 Assembly (Table 2)
      Tip: The assembled Tn5 will be used in the second tagmentation step.

      Table 2. Oligonucleotides for Tn5 assembly

      Name Oligonucleotides sequence
      Tn5MErev 5′-[phos]CTGTCTCTTATACACATCT-3′
      Tn5ME-A 5′ TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3′
      Tn5ME-B 5′ -GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′


      1. Prepare all DNA oligonucleotides (see Table 1) to 100 μM.

      2. Mix 5 µL of Tn5ME-A with 5 µL of Tn5MErev in one PCR tube, and mix 5 µL of Tn5ME-B with 5 µL of Tn5MErev in another PCR tubes. The DNA sequences of oligonucleotides are list in Table 2.

      3. Perform oligos annealing by subjecting the oligos to 95°C for 5 min followed by slow ramping to 25°C with -0.1°C/s ramping rate using a PCR machine.

      4. After that, mix all the reagents in mentioned volumes: 2 µL of Tn5MErev/Tn5ME-A, 2 µL of Tn5MErev/Tn5ME-B, 20 µL of 100% glycerol, 15.24 2 µL of 2× dialysis buffer (100 mM HEPES−KOH at pH 7.2, 0.2 M NaCl, 0.2 mM EDTA, 2 mM DTT, 0.2% Triton X-100, 20% glycerol), 2.15 µL of Tn5 (46.55 μM), 8.21 µL of water and incubate it at room temperature for 1 h (also see Table 9 for 2× dialysis buffer preparation and Table 10 for the pipetting scheme of Tn5 assembly).

    2. T7-pA-Tn5 assembly (Table 3)

      Tip: The assembled T7-pA-Tn5 is used in step C.3.d.

      Table 3. Oligonucleotides for T7-pA-Tn5 assembly

      Name Oligonucleotides sequence
      Tn5MErev 5′-[phos]CTGTCTCTTATACACATCT-3′
      T7-Tn5ME 5′-CATGAGATTAATACGACTCACTATAGGGAGAAGATGTGTATAAGAGACAG-3′

      1. Prepare all oligos (from Table 2) to 100 μM.

      2. Mix the 5 µL of T7-Tn5ME with 5 µL of Tn5MErev in a PCR tube. The DNA sequences of oligonucleotides are list in Table 3.

      3. Perform oligos annealing by subjecting the oligos to 95°C for 5 min followed by slow ramping to 25°C with -0.1°C/s ramping rate using PCR machine.

      4. After that, mix all the reagents in mentioned volumes: 4 µL of T7-Tn5ME/Tn5MErev, 20 µL of 100% glycerol, 15.58 µL of 2× dialysis buffer (100 mM HEPES−KOH at pH 7.2, 0.2 M NaCl, 0.2 mM EDTA, 2 mM DTT, 0.2% Triton X-100, 20% glycerol), 1.81 µL of pA-Tn5 (55.55 μM), 8.61 µL of water and incubate it at room temperature for 1 h (also see Table 11 for the pipetting scheme of T7 pA-Tn5 assembly and Table 8 for 2× dialysis buffer preparation).

    3. Tn5 and T7-pA-Tn5 activity assay

      Check the activity of the assembled Tn5, and T7 pA-Tn5 as mentioned in Table 4 (also see Table 12 for 2× TD buffer preparation).

      1. Mix the reagents and incubate the mixture at 55°C for 7 min. After the incubation, purify the mixture with Qiagen MiniElute PCR Purification kit according to the manufacturer protocol and eluted in 10 μL of elution buffer.

        Table 4. Mixture of Tn5 or T7-pA-Tn5 activity assay

        Reagents Volume
        2× TD buffer 10 μL
        Human genomic DNA 50 ng
        Tn5 or T7-pA-Tn5 (2 μM) 1 μL
        Water Make the final volume to 20 μL/tube

      2. Finally, mix with 2 μL of 6× loading dye and run on a 1% agarose gel to check the size of the tagmented DNA along with DNA ladder (Figure 2).

        Note: The expected size of fragments of DNA after tagmentation starts from 150 bp to 2,500 bp, and it looks like a smear spreading in the gel lane.



    Figure 2. Activity assay of Tn5, pA-Tn5, and T7-pA-Tn5.

    DNA size distribution after Tn5 transposition showed here. T7-pA-Tn5 has similar activity to Tn5 and pA-Tn5.


  3. Antibody binding and tagmentation

    1. Epitope retrieval

      1. After nuclei isolation, transfer a proper amount of FFPE nuclei (1,000–1,500,000) to a new 1.5 mL Lo-Bind tube. Then spin down the nuclei at 3,000 × g for 5 min at room temperature and discard the supernatant.

        Note: The number of nuclei used in the experiment depends on the total number of nuclei gotten from nuclei isolation step. The protocol works for 1,000 to 1,500,000 nuclei.

      2. To profile histone modification H3K27ac and H3K4me1 (euchromatin markers), resuspend the FFPE nuclei with 50 μL of Epitope Retrieval Buffer-1 (see below Table 13), and transfer the suspension to a PCR tube. Incubate the nuclei suspension at 50°C for 1 h. To profile histone modification H3K27me3 (heterochromatin marker) and H3K36me3 (gene body marker), a harsher epitope retrieval condition is needed. Therefore, the nuclei are resuspended with 50 μL of Epitope Retrieval Buffer-2 (see below Table 14), and transfer the suspension to a PCR tube. Incubate the nuclei suspension at 65°C for 1 h.

      3. During the incubation time, prepare the FACT-seq Antibody buffer (check more information from the recipe section) and keep it on ice.

      4. After incubation, add 10 μL of 10% Triton X-100 to the tube and transfer the mixture to a 1.5 mL Lo-Bind tube. And place the tube on a shaker, and incubate at 37°C for 30 min with 500 rpm to quench SDS.

      5. After quenching, spin down the mixture at 3,000 × g for 5 min at room temperature and wash the pellet once with the FACT-seq Antibody buffer.

      6. After washing, resuspend the nuclei pellet with 200 μL of FACT-seq Antibody buffer, and use 5 μL of nuclei suspension to count the nuclei number by the cell counter.

        Note: Recovery rate after epitope retrieval on average is 62.94%.

    2. Primary antibody binding

      1. After counting nuclei, transfer 100,000 FFPE nuclei into a new 0.5 mL Qubit tube.

        Note: Nuclei tend to attach to the tube wall and dry out during the overnight rotation step, and 0.5 mL tubes should be used instead of 1.5 mL tubes.

      2. Centrifuge the nuclei at 2,500 × g for 5 mins at room temperature and discard the supernatant with pipette.

      3. Resuspend the nuclei with at least 1 volume of FACT-seq Antibody buffer.

      4. Centrifuge the nuclei at 2,500 × g for 5 mins at room temperature and discard the supernatant with pipette and resuspend the nuclei with 200 μL of FACT-seq Antibody buffer with 1:100 diluted primary antibody.

      5. Incubated overnight with slow rotation (8–10 rpm) at 4°C.

        Tip: Set the rotation speed properly and don’t make the solution stuck to one side of the tube.

    3. Secondary antibody binding

      1. Prepare the following buffers before starting the experiment and keep it on ice or at +4°C.

        Tip: We always prepare these buffers freshly.

        1. FACT-seq Dig-washing buffer (see below Table 15)

        2. FACT-seq Dig-300 buffer (see below Table 16)

        3. FACT-seq tagmentation buffer (see below Table 17)

      2. After overnight incubation with primary antibody, spin down the nuclei at 2,000 × g for 5 min at room temperature, wash the nuclei pellet with 200 μL of FACT-seq Dig-washing buffer.

      3. Resuspend the nuclei pellet in 200 μL of FACT-seq Dig-washing buffer with 1:100 diluted secondary antibody (Guinea Pig anti-Rabbit IgG antibody) and incubate the mixture for 1 h at room temperature with slow rotation.

      4. Spin down the mixture at 2,000 × g for 5 min at room temperature, wash it three times with 200 μL of FACT-seq Dig-washing buffer, resuspend the nuclei pellet with 200 μL of FACT-seq Dig-300 buffer containing 1:100 diluted T7 pA-Tn5 (Tip: The T7-pA-Tn5 was assembled in section B.2.). And the protein A domain of T7 pA-Tn5 has the affinity to bind mammalian immunoglobulins, which will bind to the secondary antibody from guinea pig.

      5. Incubated the tubes for 1 h rotating at room temperature.

    4. Tagmentation and reverse crosslinking

      1. After Tn5 binding, spin down the nuclei for 5 min at 600 × g and wash three times with FACT-seq Dig-300 buffer, resuspend the nuclei with 200 μL of FACT-seq tagmentation buffer and incubate the suspension for 1 hr at 37°C.

      2. After that, stop tagmentation by addition of 6.7 μL of 0.5 M EDTA, 22 μL of 10% SDS, and 2.2 μL of 20 mg/mL Proteinase K to each tube, and mix it by gently pipetting up and down several times.

      3. Perform the Proteinase K digestion at 65°C with 1,200 rpm shaking for 2 h.

      4. Perform reverse-crosslinking overnight at 72°C with 1,200 rpm in a heating block for H3K27ac or H3K4me1 (change the overnight temperature to 80°C for H3K27me3 or H3K36me3).

      5. After overnight reverse-crosslinking, add 2.2 µL of 20 mg/mL Proteinase K and perform the Proteinase K digestion again (65°C, 1,200 rpm) for 1 h.


  4. DNA isolation and in vitro transcription

    1. DNA purification

      1. After Proteinase K digestion, purify the mixture with either Qiagen Kit purification or phenol-chloroform purification. (Tip: We don’t see differences in library quality when targeting H3K27ac using these two different purification approaches. When targeting H3K27me3 (heterochromatin marker), phenol-chloroform purification is recommended).

      2. For Qiagen purification, follow the standard protocol of Qiagen MiniElute PCR Purification kit and finally elute the DNA in 20 μL of elution buffer.

      3. For phenol-chloroform purification, after Proteinase K digestion, add elution buffer or sterile water to the tube and make the final volume 300 μL per tube. Add 300 μL of Phenol and mix by full-speed vortexing ~2 s.

      4. Centrifuge at 16,000 × g and 4°C for 15 min. Remove the aqueous layer by pipetting to a fresh 1.5 mL tube and add equal amount of Chloroform.

      5. Invert the tube ~10× to mix, and centrifuge at 16,000 × g and 4°C for 15 min.

      6. Transfer aqueous layer to a fresh 1.5 mL tube, then add 2.5×–3× volume of absolute ethanol and add proper amount of 5M NaCl to make the final concentration become 200 mM (NaCl).

      7. Transfer the tube to -80°C freezer and precipitate the DNA for 2–3 h, and centrifuge the tubes for 20 min 4 °C at 16,000 × g.

      8. Discard the supernatant and add 700 μL of 70% ethanol and slightly vortex it. Centrifuge at 16,000 × g at 4°C for 5 min, then discard the supernatant and air dry the tube for 10–15 min. After drying up, add 25 μL of elution buffer (from Qiagen MiniElution PCR Purification kit) to dissolve the DNA.

    2. Gap filling

      Tip: After Tn5 tagmentation, there will be two 9 bp gaps between the mosaic ends and the chromatin strands. And the gap filling is to fill up these two gaps using high-fidelity DNA polymerase.

      1. After DNA purification, transfer the sample to the PCR tube and add an equal volume of high-fidelity 2× PCR master mix.

      2. Then incubate the mixture for 8 mins at 72°C in a thermal cycler.

      3. Purify the sample using the Qiagen MiniElute PCR Purification kit.

    3. SPRI bead purification

      1. Remove the fragments shorter than 150 bp using 1.0× SPRI select beads. Initially, make up the volume up to 50 μL and add 1.0× SPRI, then incubate the mixture for 15 min at room temperature.

      2. After incubation, transfer the tubes to the magnetic rack for 10 min and wash the beads with 150 μL of 80% ethanol twice.

      3. After washing, let the beads dry, add 25 μL of water, incubate it for 10 min at room temperature.

      4. Transfer the tube to the magnetic rack and then carefully transfer the supernatant to a fresh tube.

    4. In vitro transcription and RNA purification

      1. Convert DNA to RNA using T7 RNA synthesis kit with 20 μL reaction system, incubate the mixture at 37 °C for overnight to get a good amount of RNA according to the manufacturer protocol.

      2. Purify the RNA using ZYMO DNase RNA purification kit and finally, elute in 15 μL of elution buffer from the kit. And measure the concentration of RNA using Nanodrop 2000c (see Table 5 for more information).

        Tip: We follow the manufactory standard protocol to perform In-vitro transcription and RNA purification. Please refer to the standard protocols from these two kits for detailed procedure.

        Table 5. Representative RNA concentration after in-vitro transcription and purification

        Initial nuclei number/tube Average RNA concentration1 (ng/μL) Total amount of RNA (μg/tube) A260/280
        50000 8898.5 133.5 2.19
        25000 8484.2 127.3 2.15
        10000 7508.4 112.6 2.11
        5000 3215.5 48.2 2.03
        2500 1012.7 15.2 1.98
        1000 139.0 2.1 1.97
        1The in-vitro transcription was performed for 22–23 h for this particular experiment


  5. Reverse transcription and pre-PCR

    1. Reverse transcription

      1. Transfer 100 ng RNA to a fresh tube, and perform reverse transcription using TAKARA reverse transcription kit. The final volume of reverse transcription is 22.2 μL/tube.

        Tip: We follow the manufactory standard protocol to perform reverse transcription. Please refer to the standard protocol from this kit for detailed procedure.

    2. cDNA purification using RNAClean XP beads

      1. make up the volume up to 40 μL and add 1.8× RNAClean XP beads, incubate the mixture for 15 min at room temperature.

        Tip: The “1.8× RNAClean XP beads” mean 1.8 times the volume. 72 μL of RNAClean XP beads is added to every 40 μL reaction.

      2. After incubation, transfer the tubes to the magnetic rack for 10 min and wash the beads with 200 μL of 70% ethanol twice.

      3. After washing, let the beads dry, add 24.2 μL of water, incubate it for 10 min, and transfer the tube to the magnetic rack. Beads bind to the magnetic rack, then carefully transfer the supernatant to a fresh tube.

    3. Pre-PCR

      1. After cDNA purification, convert single-stranded cDNA to double-stranded DNA by adding 25 μL of PCR master mix, 0.8 μL of reverse primer (25 µM) (oligonucleotides sequence is in Table 6).

      2. And then perform pre-PCR (98°C for 10 s,63°C for 30s, 72°C for 1 min, and hold at 10°C, only one cycle) and purify and elute the sample in 24.5 μL, using Qiagen MiniElute PCR Purification kit.

        Table 6. Reverse primer sequence

        Name Oligonucleotides sequence
        Reverse Primer 5′-CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGGAGATGTG-3′


  6. Second tagmentation and library preparation

    1. Second tagmentation

      1. The second tagmentation aims to add a pair of adapters to the DNA so that the DNA fragments can be amplified and sequenced properly.

      2. Perform tagmentation on the sample by adding 25 μL of 2× TD buffer (20 mM Tris-HCl pH 7.6, 10 mM MgCl2, 20% dimethyl formamide), 0.5 μL of 2 μM Tn5, and incubate at 55°C for 7 min.

      3. After incubation, purify and elute the samples in 24.2 μL of elution buffer, using Qiagen MiniElute PCR Purification kit.

    2. PCR amplification and library preparation

      1. After tagmentation and purification, perform final library amplification by adding 25 μL of 2× PCR master mix, 0.4 μL of forward primer (25 μM), 0.4 μL of reverse primer (25 μM).

        Tip: the forward and reverse primers are adapted from the 96 primer pairs in the previous report (Buenrostro et al., 2015). Please refer to the original paper for more information.

      2. Perform PCR amplification (72°C for 5 min, 20 cycles of 98°C for 10 s,63°C for 30 s, 72°C for 1 min and hold at 10°C).

      3. Purify samples with Qiagen MiniElute PCR Purification kit.

    3. Size selection and gel purification

      1. After library amplification and purification, the libraries are run on 8% acrylamide gel.

      2. Mix all of the components in 8% acrylamide gel (see Table 7. for more information) well in a clean 15 mL Falcon tube.

      3. Transfer the mixture to the corresponding polyacrylamide gel tray and let it solidify at room temperature for 30 min.

      4. During the waiting time, prepare DNA ladder and mix all of the samples with 6× DNA loading dye.

      5. Load all the samples and 50 bp DNA ladder on the gel and run at 180 V for 15–20 min.

      6. After running, transfer the gel to 1× SYBR gold and stain the gel in darkness for 20 min.

        Table 7. Recipe of 8% acrylamide gel (one gel)

        Reagents Volume (10 mL)
        40% Acrylamide:bis-acrylamide 2 mL
        10× TBE buffer 1 mL
        10% Ammonium persulfate 50 μL
        TEMED 10 μL
        Sterile Water 6.94 mL


      7. After staining, check the gel under the gel documentation system and cut each gel lane from 220–1,000 bp by referring to 50 bp DNA ladder.

      8. Transfer each gel cut into a 0.5 mL punched tube, and dip into a 2 mL Eppendorf tube. Then centrifuge both the tubes at 16,000 × g for 5 min and discard the 0.5 mL punched tube.Note: The aim of this step is to make the entire gel lane into small pieces.

      9. Add 300 μL of crush soak buffer (500 mM NaCl, 1 mM EDTA, 0.5% SDS [Table 18]) to the gel and incubate the mixture at 55°C for 8 h at least to dissolve DNA fragments in the buffer.Note: The aim of this step is to dissolve DNA fragments in the buffer.

      10. After incubation, transfer the gel mixture to costar tubes and centrifuge for 5 min at 16,000 × g.

      11. Then collect the solution passing through the costar filter and use Zymo ChIP DNA Clean and concentrator kit to purify it and finally elute in 15 μL.
        Tip: We follow the manufactory standard protocol to perform DNA purification. Please refer to the standard protocol from this kit for detailed procedure.

  1. Precise quantification and sequencing

    1. Precise quantification DNA concentration of FACT-seq libraries using Agilent high sensitivity DNA kit following manufacturing protocol.
      Tip: Please refer to the protocol from this kit for detailed procedure.

    2. Sequencing: Sequence the FACT-seq libraries on Illumina NovaSeq 6000 sequencer or Illumina MiniSeq platform with paired end sequencing, and some results (Zhao et al., 2021) are as shown in Figure 3.
      Tip: Please refer to the protocol from Illumina for detailed procedure.


      Figure 3. Representative genome browser tracks of H3K27ac sequencing libraries from mouse kidney nuclei, adopted from Zhao et al. (2021).

      Results from H3K27ac FACT-seq of mouse FFPE kidney nuclei (- epitope retrieval), H3K27ac FACT-seq of mouse FFPE kidney nuclei (+ epitope retrieval), H3K27ac CUT&Tag of frozen mouse kidney nuclei, ENCODE H3K27ac ChIP-seq of frozen mouse kidney nuclei, and CUT&Tag IgG control of mouse kidney nuclei. The gene names are shown at the bottom. Chr = Chromosome. For in detail analysis, refer to FACT-seq paper (Zhao et al., 2021).

    Data analysis

    1. Only reads one (R1) will be involved in the downstream analysis. We first adopt levenshtein distance algorithm to map T7 promoter sequence to each read in the adaptor trimming procedure; then T7 promoter sequences could be trimmed from each read in the fastq file with in-house script custom script (https://github.com/pengweixing/FACT).

    2. The trimmed fastq file of human samples and mouse samples are mapped to the hg19/hg38 reference genome or mm9/mm10 reference genome respectively using bowtie2 v.2.3.5 (Langmead and Salzberg, 2012) with the parameters --end-to-end --very-sensitive -I 10 -X 700. The aligned BAM files are sorted by samtools v.1.9 (Li et al., 2009). The duplicate reads are removed with Picard v1.79 (http://picard.sourceforge.net), and be filtered with alignment quality of >q2. For the peak calling, broad peaks could be called using SICER (Zhang et al., 2008) with the following parameters gap size = 600 bp and window size = 200 bp. Narrow peaks could be called using SICER with following parameters gap size = 400 bp and window size = 200 bp. The bigwig files were generated from BAM file using deeptools (Ramirez et al., 2014) with the parameters bamCoverage -normalizeUsing CPM. The peaks of sequencing libraries are visualized by IGV (Thorvaldsdottir et al., 2013) software.

    Notes

    1. Be extra careful when working with detergents like sodium deoxycholate, digitonin, and SDS. Remember to wear a face mask, lab coat, and gloves when dissolving the powder of sodium deoxycholate and digitonin in the liquid.
    2. We have successfully profiled H3K27ac in FFPE mouse nuclei with a limited amount of input (1,000 nuclei/tube or starting nuclei isolation from a small piece of tissue containing approximately 5,000 nuclei). So, the FACT-seq protocol could usually work even if the nuclei input is less than 100,000 nuclei/tube.
    3. Epitope retrieval condition is an important steps to obtain a good library quality. Here, we provided the best epitope retrieval conditions for H3K27ac, H3K27me3, H3K4me1, and H3K36me3. This epitope retrieval condition may directly be used for the other histone modifications, including H3K4me3, H3K9me2, or H3K9me3.
    4. FACT-seq targeting H3K27ac could be a positive control if you want to test unknown conditions. 
    5. The DNA amount we got after tagmentation and purification range from 100 ng–300 ng/tube (each tube starts from 100,000 FFPE nuclei). We would always proceed with library preparation unless the DNA concentration is too low (< 10 ng/tube) or the purity of the DNA is too bad (A260/280 < 1.5).
    6. We have successfully performed FACT-seq in FFPE human colorectal cancer and FFPE human glioblastoma samples. The RNA yield after in-vitro transcription varies when using different samples or profiling different markers. If the RNA yield of a particular sample is low, you could try extending the time in-vitro transcription from overnight (8 h) to 16 h or even longer. The longest in-vitro transcription we performed was approximately 23 h, and we still got good libraries.
    7. For RNA purification, we initially used the traditional TRIzol RNA purification and got good results. Since TRIzol purification is time-consuming, we finally switched to using the kit purification.

    Recipes

    1. Buffer used in isolation of single nuclei suspension from FFPE tissue section

      Table 8. Recipe of NST buffer (Nonidet P40 with Salts and Tris buffer)
      Reagents Initial Concentration Final Concentration Dilution Fold Final Volume (10 mL)
      NaCl 5,000 mM 146 mM 34.25 292 μL
      Tris-HCl pH 7.5 1,000 mM 5 mM 200 50 μL
      Tris-HCl pH 8 1,000 mM 5 mM 200 50 μL
      CaCl2 1,000 mM 1 mM 1000 10 μL
      MgCl2 1,000 mM 21 mM 47.62 210 μL
      BSA 10% 0.05% 200 50 μL
      IGEPAL CA-630 10% 0.20% 50 200 μL
      Nuclease-free water / / / 9138 μL

      Collagenase: 6 mg/mL in PBS with 0.5 mM CaCl2
      Hyaluronidase: 600 units/mL in PBS with 0.5 mM CaCl2

    2. Buffers used in Tn5 assembly and activity assay

      Table 9. Recipe of 2× Dialysis buffer
      Reagents Initial Concentration Final Concentration Dilution Fold Final Volume (10 mL)
      HEPES(K+) pH 7.2 500 mM 100 mM 5 2 mL
      NaCl 5,000 mM 200 mM 25 400 μL
      EDTA 500 mM 0.2 mM 2500 4 μL
      DTT 500 mM 2 mM 250 40 μL
      Triton X-100 10% 0.20% 50 200 μL
      Glycerol 100% 20% 5 2 mL
      Nuclease-free water / / / 5,356 μL

      Table 10. Recipe of Tn5 assembly
      Reagents Initial Concentration Final Concentration Dilution Fold Final Volume (50 μL)
      Tn5MErev/Tn5ME-A 50 μM 2 μM 25 2 μL
      Tn5MErev/Tn5ME-B 50 μM 2 μM 25 2 μL
      Glycerol 100% 40% 2.5 20 μL
      2× Dialysis buffer / / / 15.24 μL
      Tn5 46.55 μM 2 μM 23.26 2.15 μL
      Nuclease-free water / / / 8.61 μL

      Table 11. Recipe of T7 pA-Tn5 assembly
      Reagents Initial Concentration Final Concentration Dilution Fold Final Volume (50 μL)
      Tn5MErev/Tn5ME-A 50 μM 4 μM 12.5 4 μL
      Glycerol 100% 40% 2.5 20 μL
      2× Dialysis buffer / / / 15.58 μL
      pA-Tn5 55.55 μM 2 μM 27.76 1.81 μL
      Nuclease-free water / / / 8.61 μL

      Table 12. Recipe of 2× TD buffer
      Reagents Initial Concentration Final Concentration Dilution Fold Final Volume (10mL)
      Tris-HCl pH 7.5 1,000 mM 20 mM 50 200 μL
      MgCl2 1,000 mM 10 mM 100 100 μL
      Dimethyl formamide 100% 20% 5 2 mL
      Nuclease-free water / / / Bring up to 10 mL

    3. Buffers used in epitope retrieval

      Table 13. Recipe of Epitope Retrieval Buffer-1
      Reagents Initial Concentration Final Concentration Dilution Fold Final Volume (10 mL)
      EDTA 500 mM 10 mM 50 200 μL
      Tris-HCl pH 8 1,000 mM 50 mM 20 500 μL
      SDS 10% 0.10% 100 100 μL
      Nuclease-free water / / / 9.2 mL

      Table 14. Recipe of Epitope Retrieval Buffer-2
      Reagents Initial Concentration Final Concentration Dilution Fold Final Volume (10 mL)
      EDTA 500 mM 10 mM 50 200 μL
      Tris-HCl pH 8 1,000 mM 50 mM 20 500 μL
      SDS 10% 0.10% 100 100 μL
      Sodium deoxycholate 10% 0.10% 100 100 μL
      Nuclease-free
      water
      / / / 9.1 mL

    4. Buffers used in antibody binding and tagmentation

      Table 15. Recipe of FACT-seq Dig-washing buffer (for 8 libraries)
      Reagents Initial Concentration Final Concentration Dilution Fold Final Volume (15 mL)
      HEPES(K+) pH 7.6 500 mM 20 mM 25 600 μL
      NaCl 5,000 mM 150 mM 33.33 450 μL
      Spermidine 2,000 mM 0.5 mM 4,000 3.75 μL
      Digitonin 5% 0.05% 100 150 μL
      IGEPAL CA-630 10% 0.01% 1,000 15 μL
      BSA 10% 1% 10 1,500 μL
      Nuclease-free water
      (with protease inhibitor added)
      / / 12.28 mL

      To prepare FACT-seq Antibody buffer (for 8 libraries), transfer 4 mL of FACT-seq Dig-washing buffer to a new tube and add 16 μL of 0.5 M EDTA, mix well and keep it on ice.

      Table 16. Recipe of FACT-seq Dig-300 buffer (for 8 libraries)
      Reagents Initial Concentration Final Concentration Dilution Fold Final Volume (10 mL)
      HEPES(K+) pH 7.6 500 mM 20 mM 25 400 μL
      NaCl 5,000 mM 300 mM 16.67 600 μL
      Spermidine 2,000 mM 0.5 mM 4,000 2.5 μL
      Digitonin 5% 0.05% 100 100 μL
      IGEPAL CA-630 10% 0.01% 1,000 100 μL
      BSA 10% 1% 10 1,000 μL
      Nuclease-free water
      (with protease inhibitor added)
      / / 7.89 mL


      Table 17. Recipe of FACT-seq tagmentation buffer (for 8 libraries)
      Reagents Initial Concentration Final Concentration Dilution Fold Final Volume (2 mL)
      HEPES (K+) pH 7.6 500 mM 20 mM 25 80 μL
      NaCl 5,000 mM 300 mM 16.67 120 μL
      Spermidine 2,000 mM 0.5 mM 4,000 0.5 μL
      Digitonin 5% 0.05% 100 20 μL
      IGEPAL CA-630 10% 0.01% 1000 2 μL
      MgCl2 1,000 mM 10 mM 100 20 μL
      Nuclease-free water
      (with protease inhibitor added)
      / / 1,757.5 μL


    5. Buffer used in library preparation

      Table 18. Recipe of crush soak buffer
      Reagents Initial Concentration Final Concentration Dilution Fold Final Volume (10 mL)
      NaCl 5,000 mM 500 mM 10 1,000 μL
      EDTA 500 mM 1 mM 500 20 μL
      SDS 10% 0.50% 20 500 μL
      Nuclease-free water / / / 8,480 μL

    Acknowledgments

    This protocol is derived from the previous publication FACT-seq from Chen Lab (Zhao et al., 2021). We want to thank the contributions of all of Chen Lab’s members who involved in this work. We want to thank Protein Science Facility at Karolinska Institute for providing purified Tn5 and pA-Tn5. Our funding sources including: Swedish Research Council [VR-2016-06794, VR-201702074 to X.C.]; Åke Wibergs stiftelse [M20-0007 to X.C.];Beijer Foundation (to X.C.); Jeassons Foundation (to X.C.); Petrus och Augusta Hedlunds Stiftelse (to X.C.); Göran Gustafsson’s prize for younger researchers (to X.C.); Vleugel Foundation (to X.C.); Linnéstiftelsen for medicinsk forskning (to X.C.); Uppsala University (to X.C.); Swedish Cancer Society (CAN 2021-1449Pj, 22 0491 JIA to X.C.).

    Competing interests

    Xingqi Chen, Vamsi Krishna Polavarapu, and Linxuan Zhao have filed patent applications related to previous published FACT-seq paper and the protocol described here. The title of the patent application is “Method of preparing DNA from formalin-fixed-paraffin-embedded (FFPE) tissue samples”. The Swedish Provisional Application was filed on 28 June 2021, Patent Application No. 2150823-9 in Sweden. The authors declare no competing financial interests.

    References

    1. Astolfi, A., Urbini, M., Indio, V., Nannini, M., Genovese, C. G., Santini, D., Saponara, M., Mandrioli, A., Ercolani, G., Brandi, G., et al. (2015). Whole exome sequencing (WES) on formalin-fixed, paraffin-embedded (FFPE) tumor tissue in gastrointestinal stromal tumors (GIST). BMC Genomics 16: 892.
    2. Becker, J. S., McCarthy, R. L., Sidoli, S., Donahue, G., Kaeding, K. E., He, Z., Lin, S., Garcia, B. A. and Zaret, K. S. (2017). Genomic and Proteomic Resolution of Heterochromatin and Its Restriction of Alternate Fate Genes. Mol Cell 68(6): 1023-1037 e1015.
    3. Bolognesi, C., Forcato, C., Buson, G., Fontana, F., Mangano, C., Doffini, A., Sero, V., Lanzellotto, R., Signorini, G., Calanca, A., et al. (2016). Digital Sorting of Pure Cell Populations Enables Unambiguous Genetic Analysis of Heterogeneous Formalin-Fixed Paraffin-Embedded Tumors by Next Generation Sequencing. Sci Rep 6: 20944.
    4. Buenrostro, J. D., Wu, B., Litzenburger, U. M., Ruff, D., Gonzales, M. L., Snyder, M. P., Chang, H. Y. and Greenleaf, W. J. (2015). Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523(7561): 486-490.
    5. Cejas, P., Li, L., O'Neill, N. K., Duarte, M., Rao, P., Bowden, M., Zhou, C. W., Mendiola, M., Burgos, E., Feliu, J., et al. (2016). Chromatin immunoprecipitation from fixed clinical tissues reveals tumor-specific enhancer profiles. Nat Med 22(6): 685-691.
    6. Fanelli, M., Amatori, S., Barozzi, I. and Minucci, S. (2011). Chromatin immunoprecipitation and high-throughput sequencing from paraffin-embedded pathology tissue. Nat Protoc 6(12): 1905-1919.
    7. Fanelli, M., Amatori, S., Barozzi, I., Soncini, M., Dal Zuffo, R., Bucci, G., Capra, M., Quarto, M., Dellino, G. I., Mercurio, C., et al. (2010). Pathology tissue-chromatin immunoprecipitation, coupled with high-throughput sequencing, allows the epigenetic profiling of patient samples. Proc Natl Acad Sci U S A 107(50): 21535-21540.
    8. Font-Tello, A., Kesten, N., Xie, Y., Taing, L., Vareslija, D., Young, L. S., Hamid, A. A., Van Allen, E. M., Sweeney, C. J., Gjini, E., et al. (2020). FiTAc-seq: fixed-tissue ChIP-seq for H3K27ac profiling and super-enhancer analysis of FFPE tissues. Nat Protoc 15(8): 2503-2518.
    9. Fox, C. H., Johnson, F. .B, Whiting, J. and Roller, P. P. (1985). Formaldehyde fixation. J Histochem Cytochem 33(8): 845-53.
    10. Haile, S., Corbett, R. D., Bilobram, S., Bye, M. H., Kirk, H., Pandoh, P., Trinh, E., MacLeod, T., McDonald, H., Bala, M., et al. (2019). Sources of erroneous sequences and artifact chimeric reads in next generation sequencing of genomic DNA from formalin-fixed paraffin-embedded samples. Nucleic Acids Res 47(2): e12.
    11. Kaya-Okur, H. S., Wu, S. J., Codomo, C. A., Pledger, E. S., Bryson, T. D., Henikoff, J. G., Ahmad, K. and Henikoff, S. (2019). CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat Commun 10(1): 1930.
    12. Langmead, B. and Salzberg, S. L. (2012). Fast gapped-read alignment with Bowtie 2. Nat Methods 9(4): 357-359.
    13. Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., Abecasis, G., Durbin, R. and Genome Project Data Processing, S. (2009). The Sequence Alignment/Map format and SAMtools. Bioinformatics 25(16): 2078-2079.
    14. Pennock, N. D., Jindal, S., Horton, W., Sun, D., Narasimhan, J., Carbone, L., Fei, S. S., Searles, R., Harrington, C. A., Burchard, J., et al. (2019). RNA-seq from archival FFPE breast cancer samples: molecular pathway fidelity and novel discovery. BMC Med Genomics 12(1): 195.
    15. Picelli, S., Bjorklund, A. K., Reinius, B., Sagasser, S., Winberg, G. and Sandberg, R. (2014). Tn5 transposase and tagmentation procedures for massively scaled sequencing projects. Genome Res 24(12): 2033-2040.
    16. Ramirez, F., Dundar, F., Diehl, S., Gruning, B. A. and Manke, T. (2014). deepTools: a flexible platform for exploring deep-sequencing data. Nucleic Acids Res 42(Web Server issue): W187-191.
    17. Teytelman, L., Ozaydin, B., Zill, O., Lefrancois, P., Snyder, M., Rine, J. and Eisen, M. B. (2009). Impact of chromatin structures on DNA processing for genomic analyses. PLoS One 4(8): e6700.
    18. Thorvaldsdottir, H., Robinson, J. T. and Mesirov, J. P. (2013). Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform 14(2): 178-192.
    19. Waldron, L., Simpson, P., Parmigiani, G. and Huttenhower, C. (2012). Report on emerging technologies for translational bioinformatics: a symposium on gene expression profiling for archival tissues. BMC Cancer 12: 124.
    20. Wang, Y., Moorhead, M., Karlin-Neumann, G., Falkowski, M., Chen, C., Siddiqui, F., Davis, R. W., Willis, T. D. and Faham, M. (2005). Allele quantification using molecular inversion probes (MIP). Nucleic Acids Res 33(21): e183.
    21. Zhang, Y., Liu, T., Meyer, C. A., Eeckhoute, J., Johnson, D. S., Bernstein, B. E., Nusbaum, C., Myers, R. M., Brown, M., Li, W., et al. (2008). Model-based analysis of ChIP-Seq (MACS). Genome Biol 9(9): R137.
    22. Zhao, L., Xing, P., Polavarapu, V. K., Zhao, M., Valero-Martinez, B., Dang, Y., Maturi, N., Mathot, L., Neves, I., Yildirim, I., et al. (2021). FACT-seq: profiling histone modifications in formalin-fixed paraffin-embedded samples with low cell numbers. Nucleic Acids Res 49(21): e125.

简介

基础研究和转化癌症研究中的大多数活检都以存档的福尔马林固定石蜡包埋 (FFPE) 样本的形式保存。分析存档 FFPE 组织中的组蛋白修饰对于了解人类疾病中的基因调控至关重要。当前来自 FFPE 样本的全基因组组蛋白修饰分析研究所需的输入是 10-20 个组织切片或整个组织块,这阻碍了更好的解析分析。然而,由于感兴趣的临床组织是有限的,因此在分析中需要消耗最少量的 FFPE 组织切片。在这里,我们展示了具有抗体引导的染色质标记和测序 (FACT-seq) 的 FFPE 组织,这是一种通过结合高活性 Tn5 转座酶和蛋白 A 的新型融合蛋白 (T7-pA-Tn5) 来有效分析 FFPE 组织中组蛋白修饰的高灵敏度方法。 ) 转座和 T7 体外转录。 FACT-seq 从具有少量 FFPE 核的不同组蛋白修饰生成高质量的染色质图谱。我们展示了一小块含有约 4000 个核的 FFPE 组织切片,足以用 FACT-seq 解码 H3K27ac 修饰。在存档的 FFPE 人类结肠直肠癌和人类胶质母细胞瘤癌组织中,H3K27ac FACT-seq 揭示了疾病特异性超级增强剂。总之,FACT-seq 允许研究人员以高灵敏度解码存档 FFPE 组织中的 H3K27ac 和 H3K27me3 等组蛋白修饰,从而使我们能够了解表观遗传调控。


图形概要:



( i ) FFPE 组织切片; ( ii ) 孤立的原子核; ( iii ) 一抗、二抗和T7-pA-Tn5与靶标结合; ( iv ) DNA 纯化; ( v )体外转录和测序文库制备; (六) 测序


背景

目前,福尔马林固定石蜡包埋 (FFPE) 是基础研究和转化研究中活检标本处理的通用方法( Fox等人,1985;Wang等人,2005; Haile等人,2019 )。此外,据报道,全球每年都有大量 FFPE 标本存档( Waldron等人,2012 年)。已经开发了越来越多的技术来破译基因组( Astolfi 等人,2015;博洛涅西 FFPE 样本中的转录组学( Pennock等人,2019 年)和蛋白质组学( Becker等人, 2017 年)信息。然而,缺乏一种能够以高灵敏度分析 FFPE 样品中的表观遗传调控的方法。此前,研究人员将染色质免疫沉淀和测序 ( ChIP -seq) 应用于存档的 FFPE 组织。并成功开发了病理组织染色质免疫沉淀(PAT - ChIP )( Fanelli et al ., 2010, 2011 )、固定组织染色质免疫沉淀测序( FiT -seq)( Cejas ) 等人,2016 年)和用于 H3K27 乙酰化分析的固定组织ChIP -seq ( FiTAc - seq)( Font-Tello等人,2020 年)使得分析 FFPE 组织中的组蛋白修饰成为可能。然而,这些技术需要来自 FFPE 样本的输入是 10-20 个组织切片或整个组织块。由于临床样本通常有限,因此较高的样本消耗量会阻止它们在更广泛的应用中使用。此外,这些可用方法中包括超声处理,这可能会引入测序偏差( Teytelman等人,2009 年)。在这里,我们 提出了一种新的高灵敏度方法,我们最近开发了该方法,使用抗体引导的染色质标记和测序 (FACT-seq) 有效地分析 FFPE 样品中的组蛋白修饰( Zhao et al ., 2021 )。 FACT-seq 使用一种新型融合蛋白 (T7-Pa-Tn5),将高活性 Tn5 转座酶与蛋白 A 结合,利用 T7体外转录制备文库,并在少量 FFPE 样品中分析组蛋白修饰。

关键字:FFPE组织, 组蛋白修饰, 高灵敏度, T7-pA-Tn5 转座酶, FACT-seq

材料和试剂
1. 50 mL Falcon管( Sarstedt ,目录号: 62.559.001)
2. 21 G针(BD Microlance ,目录号: ND432)
3. 27 G针(BD Microlance ,目录号: 302200)
4. 1 mL注射器(BD Microlance ,目录号: 309628)
5. 1.5 mL低结合管( Sarstedt ,目录号:72.706.600)
6. 0.5 mL Qubit 管(Invitrogen,目录号: Q32856)
7. 30 μm过滤器( Miltenyi Biotech MACS,目录号:130-098-458)(室温)
8. 二甲苯( Histolab ,目录号:2070)(室温)
9. 乙醇(VWR BDH Chemicals,目录号:VWRC20816.552)(室温)
10. 胶原酶(Sigma-Aldrich,目录号:C9263)(-20°C)
11. 透明质酸酶(Merck Millipore,目录号:HX0154)(-20°C)
12. 氨苄青霉素( Serva ,目录号:69-52-3)(+4°C)
13. 叠氮化钠( Merck Millipore,目录号:822335)(室温)
14. BSA( Miltenyi Biotech MACS,目录号:130-091-376)(+4°C)
15. IGEPAL CA-630(Sigma-Aldrich,目录号:I3021)(+4°C)
16. 1 M CaCl 2 (Alfa Aesar ,目录号:J63122)(室温)
17. 5 M NaCl(Thermo Fisher Scientific,Invitrogen,目录号:AM9759)(室温)
18. 1 M Tris-HCl pH 8.0(Thermo Fisher Scientific,Invitrogen,目录号:15568-025)
19. 1 M Tris-HCl pH 7.5(Thermo Fisher Scientific,Invitrogen,目录号: 15567-027 )
20. 1 M MgCl 2 (Thermo Fisher Scientific,Invitrogen,目录号:AM9530G)(室温)
21. FBS(Life Technologies,目录号:10108-105)(-20 °C )
22. RNase A(Thermo Fisher Scientific,目录号:EN0531)(- 20 °C)
23. DTT(Thermo Fisher Scientific,目录号:20291)(+4°C)
24. 0.5 M EDTA(Thermo Fisher Scientific,Invitrogen,目录号:AM9260G)(室温)
25. 二甲基甲酰胺(Sigma-Aldrich,目录号:D4551)(室温)
26. 甘油(Sigma-Aldrich,目录号:G9012)(室温)
27. 脱氧胆酸钠(Sigma-Aldrich,目录号:D6750)(室温)
28. Triton X-100(Sigma-Aldrich,目录号:T8787)(室温)
29. 人类基因组 DNA(Promega,目录号:G3041)(+4°C)
30. Qiagen miniElute PCR 纯化试剂盒(Qiagen,目录号:28004)
31. 6X加载染料(Thermo Fisher Scientific,目录号:R0611)(-20°C)
32. HEPES(Sigma-Aldrich,目录号:H3375)
33. 蛋白酶抑制剂(Sigma-Aldrich,目录号:11873580001)
34. 亚精胺(Sigma-Aldrich,目录号:S2626)
35. 10% SDS( Thermo Fisher Scientific, Invitrogen,目录号: 1553-035 )
36. 抗H3K27ac抗体(Abcam,目录号:ab4729)
37. 抗 H3K27me3 抗体(Cell Signaling Technology,目录号:9733S)
38. 抗H3K4me1抗体(Abcam,目录号:ab176877)
39. 抗H3K36me3抗体(Abcam,目录号:ab9050)
40. 小鼠 IgG1,κ 单克隆(Abcam,目录号:ab18443)
41. 豚鼠抗兔IgG抗体(Antibodies-Online,目录号:ABIN101961)
42. 蛋白酶K(Thermo Fisher Scientific,目录号: EO0491)
43. 苯酚(Thermo Fisher Scientific,目录号: 17914)
44. 氯仿(Sigma-Aldrich,目录号: C2432)
45. NEBNext高保真 2 × PCR 预混液(New England Biolabs,目录号:M0541S)(-20°C)
46. SPRIselect珠子(Beckman Coulter,目录号:B23317)(室温)
47. T7高产量RNA合成试剂盒(New England Biolabs,目录号:E2040S)(-20°C)
48. Zymo RNA纯化试剂盒( Zymo Research ,目录号:R1013)(室温)
49. SMART MMLV 套件(TAKARA,目录号:639524)(-20°C)
50. RNAClean XP 珠子(Beckman Coulter,目录号:A63987)(+4°C)
51. 齐莫 ChIP DNA 清洁和浓缩试剂盒( Zymo Research,目录号: D5205) (室温)
52. 40%丙烯酰胺:双-丙烯酰胺(Invitrogen,目录号:HC2040)
53. 10%过硫酸铵(Invitrogen,目录号:HC2005)
54. TEMED(Invitrogen,目录号:HC2006)
55. Digitonin ( Millipore ,目录号: 300410 )
56. 无核酸酶水(Invitrogen,目录号:AM9932 )
57. KOH(Sigma-Aldrich,目录号: 484016)
58. 50 bp DNA 梯( ThermoFisher Scientific,目录号:10488099)
59. 安捷伦高灵敏度 DNA 试剂盒(安捷伦,目录号:5067-4626)
60. Tn5 转座酶 [根据先前的描述在当地蛋白质设施中生产( Picelli 等人,2014 年)]
61. pA-Tn5 转座酶 [根据先前的描述在当地蛋白质设施中生产( Picelli 等 等,2019 年)]
62. 1 × PBS(pH = 7.4)( Thermo Fisher Scientific,目录号: 10010023 )
63. SYBR 金( ThermoFisher Scientific,目录号:S33102)
64. 10 × TBE缓冲液( ThermoFisher Scientific,目录号:B52)
65. 小鼠肾脏/肝脏 FFPE 组织块(实验室制备)
66. 用于从 FFPE 组织中分离单核悬浮液的缓冲液(参见配方)
67. Tn5 组装和活性测定中使用的缓冲液(参见食谱)
68. 用于表位检索的缓冲液(参见配方)
69. 用于抗体结合和标记的缓冲液(参见配方)
70. 用于文库制备的缓冲液(参见配方)




设备


1. 切片机( Leica, Histo Core MULTICUT 半自动旋转切片机,目录号:149MULTI0C1、14051856372 或类似的)
2. 立体显微镜( Zeiss stemi DV4立体显微镜8x-32x,目录号:435421-0000-000或类似的)
3. ThermoMixer ( Eppendorf,Thermomixer F1.5,目录号:5385000016 或类似产品)
4. 热循环仪( Applied Biosystems, Veriti ,目录号:4375786或类似的热循环仪)
5. NanoDrop ( Thermo Fisher Scientific ,Nanodrop 2000c,目录号: ND2000CLAPTOP或类似产品)
6. 细胞计数器( Life Technologies,Countess II,目录号:AMQAF1000 或类似产品)
7. 离心机( Eppendorf centrifuge 5415R,目录号:EP5415R 或类似的)
8. 旋转器( Grant Instruments TM 360° Vertical Multi-function Rotator PTR 35 ,目录号:9.721028或类似产品)
9. 显微镜( Zeiss Imager.Z2,目录号:490016-0001-000 或类似的)
10. DynaMag TM -2 磁架( Thermo Fisher Scientific ,目录号:12321D 或类似的)
11. 安捷伦 DNA 生物分析仪(安捷伦,目录号: 5067-4626)
12. 聚丙烯酰胺凝胶托盘(Thermo Fisher Scientific ,目录号: HC1000S)
13. 凝胶文档系统(BIO-RAD,目录号:1708195EDU)




软件


1. 领结2( http://bowtie-bio.sourceforge.net/bowtie2/index.shtmL )
2. Samtools ( http://samtools.sourceforge.net/ )
3. 深度工具( https://deeptools.readthedocs.io/en/develop/content/installation.htmL )
4. SICER ( https://zanglab.github.io/SICER2/ )
5. Picard 工具 ( https://broadinstitute.github.io/picard/ )
6. IGV ( https://software.broadinstitute.org/software/igv/ )
7. 斐济( https://imagej.net/software/fiji/ )




程序


A. 从 FFPE 块中分离单核悬浮液
1. 将 FFPE 块切成小块或卷曲
2. 脱蜡和再水化
3. 总组织碎裂
4. 分解
5. 过滤
B. TN5组装
1. TN5组装
2. T7-pA-Tn5 组件
3. Tn5 和 T7-pA-Tn5 活性测定
C. 抗体结合和标记
1. 表位检索
2. 一抗结合
3. 二抗结合
4. 标记和反向交联
D. DNA分离和体外转录
1. DNA纯化
2. 间隙填充
3. SPRI 珠纯化
4. 体外转录和 RNA 纯化
E. 逆转录和预PCR
1. 逆转录
2. 使用RNAClean纯化 cDNA XP珠
3. PCR前
F. 第二次标记和文库制备
1. 二次标记
2. PCR扩增
3. 尺寸选择和凝胶纯化
G. 精确定量和测序
1. 使用生物分析仪进行精确定量
2. 测序




A. 从 FFPE 组织块中分离单核悬浮液


FFPE 组织通过多个步骤进行处理:脱蜡、再水化、总组织破碎、酶消化、解聚和过滤以分离单核悬浮液。
1. 将 FFPE 块切片(或卷曲或幻灯片)并制备酶。
以不同的厚度对 FFPE 块进行切片:7 – 20 μm 。
提示:为了获得良好的切片而没有任何组织破损,切片前用湿乙醇无菌布擦拭,在组织块顶部添加一些乙醇。
2. 脱蜡和再水化
a. Falcon 管中取一个组织切片,加入 1 mL 二甲苯进行脱蜡,孵育 5 分钟。孵化后,吸出二甲苯并重复相同的步骤两次。
b. 脱蜡后,加入 1 mL 的 100% 乙醇并孵育 5 分钟,然后通过添加和吸入不同百分比的乙醇(1 mL)(95%、70%、50% 和 30%)连续补液。最后,加入 1 mL 的无核酸酶水进行补液并孵育 5 分钟。
3. 组织显微切割和酶消化
a. 补液后,将组织重新悬浮在 1 mL 的 PBS(室温)中,加入 0.5 mM CaCl 2 ,选择切片,然后使用镊子将组织转移到装有 200 μL PBS 和 0.5 mM CaCl 2的高压釜玻璃盘中。在立体显微镜下使用两个 21 G 针头进行总组织碎裂。
提示:要获得合适的细胞核,请使组织尽可能小。
b. 将碎片组织转移到带有 1 mL 移液器的 1.5 mL 管中,并在 3,000 × g (室温)下将样品离心10 分钟。离心后,吸出上清液,加入 500 μL 的 6 mg/mL 胶原酶(在 PBS 中用 0.5 mM CaCl 2稀释)、500 μL 的 600 unit/mL 透明质酸酶(在 PBS 中用 0.5 mM CaCl 2稀释)、50 μg钠叠氮化物(Merck Millipore, 26628-22-8),100 μg氨苄青霉素并在 37°C 下孵育 16 小时。
4. 分解
a. 准备新鲜的NST 缓冲液( N onidet P40 与S alts 和Tris缓冲液)(NST 缓冲液配方参见表 8)。孵育 16 小时后,加入 400 μL NST 缓冲液并以 3,000 × g的速度将混合物离心10 分钟(室温),然后吸出并丢弃上清液。
b. 将 800 μL NST 缓冲液、10% 胎牛血清(88 μL )(冰上)和 0.1% DNase-free RNase A (0.88 μL )(冰上)加入颗粒中,并通过 2 个 7G 针头注射器至少分解 30 次。
5. 过滤
a. 分解后,将混合物以 3,000 × g离心10 分钟,吸出上清液,加入 800 μL NST 缓冲液,并使用 30 μm 过滤器过滤混合物两次。
b. 过滤后的混合物应以 3,000 × g离心10 分钟。然后,先弃上清,将沉淀重悬于 500 μL 1 × PBS 中,取 5 μL细胞核悬液用细胞计数器计数,取 20 μL细胞核悬液用 DAPI 染色细胞核,并可视化单个核群(图 1)。
注意: 500,000–1,000 000 个细胞核可以从一个 20 μm厚的小鼠肾脏切片中获得。




 


图 1. 分离的 FFPE 小鼠细胞核。
从 20 µm 厚的小鼠 FFPE 肾组织切片中分离出的代表性细胞核并用 DAPI 染色(比例尺 = 10 µm)。


B. TN5组装


Tn5 组装主要包括将 Tn5 适配器寡核苷酸加载到 Tn5 或 pA-Tn5 转座酶上并检查 Tn5 和 T7-pA-Tn5 活性的步骤(见表 1)。


表 1.组装 Tn5 的总结


姓名 已加载适配器
Tn5 Tn5ME-A/Tn5MErev + Tn5ME-B/Tn5MErev
T7-PA-Tn5 T7-Tn5ME/Tn5MErev


提示:开始实验前准备 2 ×透析缓冲液、Tn5 和 T7-pA-Tn5。


1. Tn5 组件(表 2)
提示:组装的 Tn5 将用于第二个标记步骤。


表 2.用于 Tn5 组装的寡核苷酸


姓名 寡核苷酸序列
Tn5MErev 5′-[磷酸] CTGTCTCTTATACACATCT -3′
TN5ME-A 5' TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3'
TN5ME-B 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3'


a. 将所有 DNA 寡核苷酸(见表 1)制备至 100 μM 。
b. 在一个 PCR管中混合 5 μL的 Tn5ME- A和 5 μL 的 Tn5MErev,在另一个 PCR 管中混合 5 μL的 Tn5ME-B 和 5 μL 的 Tn5MErev 。寡核苷酸的DNA序列 列于表 2。
c. 进行寡核苷酸退火,然后使用 PCR 机器以 -0.1°C/s 的升温速率缓慢升温至 25°C。
d. 之后,混合上述体积中的所有试剂:2 µL Tn5MErev/Tn5ME-A,2 µL Tn5MErev/Tn5ME-B, 20 µL 100% 甘油,15.24 2 µL 2 ×透析缓冲液(100 mM HEPES-KOH,pH 7.2,0.2 M NaCl,0.2 mM EDTA,2 mM DTT,0.2% Triton X-100,20% 甘油),2.15 µL Tn5 (46.55 μM ), 8.21 μL 水并在室温下孵育 1 小时(另请参阅表 9 了解 2 ×透析缓冲液的制备和表 10 了解 Tn5 组件的移液方案)。


2. T7-pA-Tn5 组件(表 3)
提示:组装的 T7-pA-Tn5 用于步骤 C.3.d。


表 3.用于T7-pA-Tn5组装的寡核苷酸


姓名 寡核苷酸序列
Tn5MErev 5′-[磷酸] CTGTCTCTTATACACATCT -3′
T7-Tn5ME 5'-CATGAGATTAATACGACTCACTATAGGGAGAAGATGTGTATAAGAGACAG-3'


a. 将所有寡核苷酸(来自表 2)准备到 100 μM 。
e. 混合 5 μL 5 µL 的 T7-Tn5ME 在 PCR 管中的 Tn5MErev。寡核苷酸的DNA序列 列于表 3。
b. 通过将寡核苷酸置于 95°C 下 5 分钟进行寡核苷酸退火,然后使用 PCR 机器以 -0.1°C/s 的升温速率缓慢升温至 25°C。
c. 之后,混合上述体积中的所有试剂:4 µL T7-Tn5ME/Tn5MErev,20 µL 100% 甘油,15.58 µL 2 ×透析缓冲液(100 mM HEPES-KOH,pH 7.2,0.2 M NaCl,0.2 mM EDTA,2 mM DTT,0.2% Triton X-100, 20% 甘油),1.81 µL pA-Tn5 (55.55 μM ), 8.61 μL 水并在室温下孵育 1 小时(T7 pA-Tn5 组件的移液方案参见表 11,2 ×透析缓冲液制备参见表 8)。
3. Tn5 和 T7-pA-Tn5 活性测定
检查组装的 Tn5 和 T7 pA-Tn5 的活性,如表 4 中所述(另见表 12 以了解 2 × TD 缓冲液的制备)。
a. 在 55°C下孵育混合物7 分钟。孵育后,根据制造商的方案用 Qiagen MiniElute PCR 纯化试剂盒纯化混合物,并在 10 μL洗脱缓冲液中洗脱。

表 4. Tn5 或 T7-pA-Tn5 活性测定的混合物


试剂 体积
2 × TD 缓冲器 10微升
人类基因组 DNA 50 纳克
Tn5 或 T7-pA-Tn5 (2 μM ) 1微升
水 使最终体积为 20 μL /管


b. 最后,与 2 μL的 6 ×上样染料混合并在 1% 琼脂糖凝胶上运行,以检查标记 DNA 的大小以及DNA 阶梯(图 2)。
注意:标记后 DNA 片段的预期大小从 150 bp 到 2,500 bp开始,并且看起来像在凝胶泳道中扩散的涂片。




 


图 2. Tn5、pA-Tn5 和 T7-pA-Tn5 的活性测定。 
此处显示了 Tn5 转座后的 DNA 大小分布。 T7-pA-Tn5 与 Tn5 和 pA-Tn5 具有相似的活性。


C. 抗体结合和标记


1. 表位检索
a. 核分离后,将适量的 FFPE 核 (1,000–1,500,000) 转移到新的 1.5 mL Lo-Bind 管中。然后在室温下以 3,000 × g离心 5 分钟并弃去上清液。
注意:实验中使用的核数取决于从核分离步骤中获得的核总数。该协议适用于 1,000 到 1,500,000 个原子核。
b. 要分析组蛋白修饰 H3K27ac 和 H3K4me1(常染色质标记),用 50 μL Epitope Retrieval Buffer-1(见下表 13)重悬 FFPE 核,并将悬浮液转移到 PCR 管中。在 50 °C 下孵育核悬浮液1 小时。要分析组蛋白修饰 H3K27me3(异染色质标记)和 H3K36me3(基因体标记),需要更苛刻的表位检索条件。因此,用 50 μL Epitope Retrieval Buffer-2 (见下表 14 )重悬细胞核,并将悬浮液转移到 PCR 管中。在 65 °C 下孵育核悬浮液1 小时。
c. 在孵育期间,准备 FACT-seq 抗体缓冲液(查看配方部分的更多信息)并将其置于冰上。
d. 孵育后,将 10 μL的10% Triton X-100 添加到管中,并将混合物转移到 1.5 mL Lo-Bind 管中。将试管置于摇床上,在 37 °C下以 500 rpm 的转速孵育 30 分钟以淬灭 SDS。
e. 淬灭后,在室温下以 3,000 × g离心5 分钟,然后用 FACT-seq Antibody 缓冲液清洗沉淀一次。
f. 洗涤后,用 200 μL FACT-seq Antibody 缓冲液重悬细胞核颗粒,并使用 5 μL细胞核悬浮液通过细胞计数器计数细胞核数。
注:表位修复后平均回收率为62.94%。
2. 一抗结合
a. 计数核后,将 100,000 个 FFPE 核转移到新的 0.5 mL Qubit 管中。
注意:在隔夜旋转步骤中,细胞核往往会附着在管壁上并变干,应使用 0.5 mL 管而不是 1.5 mL 管。
b. 在室温下以 2,500 × g离心细胞核5 分钟,然后用移液器弃去上清液。
c. 用至少 1 体积的 FACT-seq 抗体缓冲液重悬细胞核。
d. 在室温下以 2,500 × g将细胞核离心 5 分钟,然后用移液器弃去上清液,并用 200 μL FACT-seq Antibody 缓冲液和 1:100 稀释的一抗重悬细胞核。
e. 在 4°C 下缓慢旋转 (8–10 rpm) 孵育过夜。
提示:适当设置转速,不要让溶液粘在管子的一侧。
3. 二抗结合
a. 在开始实验之前准备以下缓冲液,并将其保存在冰上或 +4°C 下。
提示:我们总是新鲜制备这些缓冲液。
i. FACT-seq Dig-洗涤缓冲液(见下表 15)
ii. FACT-seq Dig-300 缓冲液(见下表 16)
iii. FACT-seq标记缓冲液(见下表 17)
b. 与一抗孵育过夜后,在室温下以 2,000 × g离心 5 分钟,用 200 μL FACT-seq Dig-washing 缓冲液清洗细胞核颗粒。
c. 在 200 μL的 FACT-seq Dig 洗涤缓冲液中重新悬浮细胞核颗粒,并在室温下缓慢旋转孵育混合物 1 小时。
d. × g的速度旋转混合物 室温下 5 分钟,用 200 μL FACT-seq Dig-washing 缓冲液洗涤 3 次,用 200 μL含有 1:100 稀释 T7 pA-Tn5的 FACT-seq Dig-300 缓冲液重悬细胞核沉淀(提示: T7-pA-Tn5 在 B.2 节中组装。 ) 。并且T7 pA-Tn5的蛋白A结构域具有与哺乳动物免疫球蛋白结合的亲和力,该免疫球蛋白将与豚鼠的二抗结合。
e. 将试管在室温下旋转 1 小时。
4. 标记和反向交联
a. × g离心 5 分钟并用 FACT-seq Dig-300 缓冲液洗涤 3 次,用 200 μL FACT-seq标记缓冲液重悬细胞核,并在 37 °下孵育悬浮液 1小时C。
b. 之后,通过在每个管中加入 6.7 μL的0.5 M EDTA、22 μL的 10% SDS 和 2.2 μL的 20 mg/mL 蛋白酶 K 来停止标记,并通过轻轻上下吹打几次来混合。
c. °C下进行蛋白酶 K 消化,以 1,200 rpm 摇动 2 小时。
d. °C 下过夜进行反向交联 在 H3K27ac 或 H3K4me1 的加热块中转速为 1,200 rpm(将 H3K27me3 或 H3K36me3 的过夜温度更改为80 °C)。
e. 过夜反向交联后,加入 2.2 µL 的 20 mg/mL 蛋白酶 K,再次进行蛋白酶 K 消化(65°C,1,200 rpm)1 小时。


D. DNA分离和体外转录


1. DNA纯化
a. 蛋白酶 K 消化后,使用 Qiagen 试剂盒纯化或苯酚-氯仿纯化对混合物进行纯化。 (提示:使用这两种不同的纯化方法靶向 H3K27ac 时,我们没有发现文库质量存在差异。当靶向 H3K27me3(异染色质标记)时,建议使用苯酚-氯仿纯化)。
b. 对于 Qiagen 纯化,遵循Qiagen MiniElute PCR Purification kit的标准方案,最后在 20 μL洗脱缓冲液中洗脱 DNA。
c. 对于苯酚-氯仿纯化,蛋白酶 K消化后,向管中加入洗脱缓冲液或无菌水,使每管终体积为 300 μL 。加入 300 μL苯酚并全速混合 涡旋约 2 秒。
d. 16,000 × g和 4°C 离心 15 分钟。通过移液到新的 1.5 mL 管中除去水层,并加入等量的氯仿。
e. 将试管倒置约 10 ×以混合,并在 16,000 × g和 4°C 下离心 15 分钟。
f. 将水层转移到新的 1.5 mL 试管中,然后加入 2.5 × –3 ×体积的无水乙醇和适量的 5M NaCl,使终浓度变为 200 mM (NaCl)。
g. 将试管转移至 -80 °C冰箱并沉淀 DNA 2-3 小时,然后在 16,000 和 4 °C 下将试管离心 20 分钟 × 克。
h. 弃去上清液,加入 700 μL的 70% 乙醇并轻轻涡旋。 16,000离心机 × g在 4°C 下保持 5 分钟,然后弃去上清液并将管风干 10-15 分钟。干燥后,加入 25 μL洗脱缓冲液(来自 Qiagen MiniElution PCR Purification kit)以溶解 DNA。
2. 间隙填充
提示:Tn5 标记后,马赛克末端和染色质链之间会有两个 9 bp 的间隙。而填空就是用高保真DNA聚合酶来填补这两个空缺。
a. DNA 纯化后,将样品转移到 PCR 管中,加入等体积的高保真2 × PCR 预混液。
b. 然后将混合物在热循环仪中于 72°C 孵育 8 分钟。
c. 使用 Qiagen MiniElute PCR 纯化试剂盒纯化样品。
3. SPRI 珠纯化
a. × SPRI 选择珠去除小于 150 bp 的片段。最初,将体积补足至 50 μL并加入 1.0 × SPRI ,然后将混合物在室温下孵育 15 分钟。
b. 孵化后,将管子转移到磁架上 10 分钟,然后用 150 μL的 80% 乙醇清洗珠子两次。
c. 洗涤后,让磁珠干燥,加入 25 μL水,室温孵育 10 分钟。
d. 将试管转移到磁架上,然后小心地将上清液转移到新试管中。
4. 体外转录和 RNA 纯化
a. μL反应系统的T7 RNA 合成试剂盒将 DNA 转化为 RNA,根据制造商的方案将混合物在 37°C 下孵育过夜,以获得大量的 RNA 。
b. 使用 ZYMO DNase RNA 纯化试剂盒纯化 RNA,最后用试剂盒中的 15 μL洗脱缓冲液洗脱。并使用 Nanodrop 2000c 测量 RNA 浓度(更多信息参见表 5)。
提示:我们遵循制造商标准方案进行体外转录和 RNA 纯化。有关详细程序,请参阅这两个套件的标准协议。


表 5.体外转录和纯化后的代表性 RNA 浓度
初始核数/管 平均 RNA 浓度1 (ng/ μ L ) RNA总量(微克/管) 260/280 _
50000 8898.5 133.5 2.19
25000 8484.2 127.3 2.15
10000 7508.4 112.6 2.11
5000 3215.5 48.2 2.03
2500 1012.7 15.2 1.98
1000 139.0 2.1 1.97
1对于这个特定的实验,体外转录进行了 22 – 23 小时


E. 逆转录和预PCR


1. 逆转录
a. 将 100 ng RNA 转移到新试管中,并使用 TAKARA 逆转录试剂盒进行逆转录。逆转录终体积为 22.2 μL /管。
提示:我们按照制造商标准协议进行逆转录。有关详细程序,请参阅此套件中的标准协议。
2. g RNAClean XP 微珠纯化 cDNA
a. 补足体积至 40 μL并加入 1.8 × RNAClean XP 珠子,在室温下孵育混合物 15 分钟。
提示:“1.8 × RNAClean XP 微珠”是指体积的 1.8 倍。每 40 μL反应液中加入72 μL RNAClean XP 微珠。
b. 孵化后,将管子转移到磁架上 10 分钟,然后用 200 μL的 70% 乙醇清洗珠子两次。
c. 洗涤后,让磁珠干燥,加入 24.2 μL水,孵育 10 分钟,然后将试管转移到磁力架上。珠子与磁架结合,然后小心地将上清液转移到新试管中。
3. PCR前
a. μL PCR 预混液、0.8 μL反向引物( 25 μM )(寡核苷酸序列见表 6) ,将单链 cDNA 转化为双链 DNA 。
b. 然后进行 pre-PCR(98°C 10 s,63°C 30s,72°C 1 min,10°C 保持,仅一个循环)并使用 Qiagen纯化和洗脱 24.5 μL样品MiniElute PCR 纯化试剂盒。


表 6.反向引物序列


姓名 寡核苷酸序列
反向底漆 5'- CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGGAGATGTG -3'


F. 第二次标记和文库制备


1. 二次标记
a. 第二个标记旨在向 DNA 添加一对接头,以便 DNA 片段可以正确扩增和测序。
b. 通过添加 25 μL的 2 × TD 缓冲液(20 mM Tris-HCl pH 7.6、10 mM MgCl 2 、 20% 二甲基甲酰胺)、0.5 μL的 2 μM Tn5 对样品进行标记,并在 55°C 下孵育 7 分钟.
c. ,使用 Qiagen MiniElute PCR 纯化试剂盒在 24.2 μL洗脱缓冲液中纯化和洗脱样品。
2. PCR扩增和文库制备
a. 标记和纯化后,通过添加 25 μL 2 × PCR 预混液、0.4 μL正向引物 (25 μM )、0.4 μL反向引物 (25 μM )进行最终文库扩增。
提示:正向和反向引物改编自之前报告中的 96 对引物 ( Buenrostro et al., 2015 )。请参阅原始论文以获取更多信息。
b. 进行 PCR 扩增(72°C 5 分钟,20 个循环,98°C 10 秒,63°C 30 秒,72°C 1 分钟并保持在 10°C)。
c. 使用 Qiagen MiniElute PCR 纯化试剂盒纯化样品。
3. 尺寸选择和凝胶纯化
a. 文库扩增和纯化后,文库在 8% 丙烯酰胺凝胶上运行。
b. 充分混合8%丙烯酰胺凝胶(参见表 7. 了解更多信息)中的所有组分。
c. 将混合物转移到相应的聚丙烯酰胺凝胶托盘中,使其在室温下固化 30 分钟。
d. 在等待期间,准备 DNA 梯并将所有样品与 6 × DNA 上样染料混合。
e. 将所有样品和 50 bp DNA 梯加载到凝胶上,并在 180 V 下运行15-20分钟。
f. 运行后,将凝胶转移到 1 × SYBR gold 中并在黑暗中染色 20 分钟。


表 7 。 8%丙烯酰胺凝胶(一凝胶)配方


试剂 体积(10 毫升)
40%丙烯酰胺:双丙烯酰胺 2 毫升
10× TBE 缓冲液 1毫升
10% 过硫酸铵 50微升
TEMED 10微升
无菌水 6.94 毫升


g. 染色后,在凝胶记录系统下检查凝胶,并参考 50 bp DNA 梯从 220-1,000 bp切割每个凝胶泳道。
h. 将每个凝胶切入 0.5 mL 冲孔管中,然后浸入 2 mL Eppendorf 管中。然后将两个管以 16,000 × g离心5 分钟,并丢弃 0.5 mL 穿孔管。
注意:此步骤的目的是将整个凝胶通道变成小块。
i. 添加 300 μL 将粉碎浸泡缓冲液(500 mM NaCl、1 mM EDTA、0.5% SDS [表 18])加入凝胶中,并在 55°C 下孵育混合物至少 8 小时以溶解缓冲液中的 DNA 片段。
注意:此步骤的目的是溶解缓冲液中的 DNA 片段。
j. × g离心 5 分钟。
k. 然后收集通过 costar 过滤器的溶液并使用Zymo ChIP DNA Clean and concentrator 试剂盒对其进行纯化,最后在 15 μL中洗脱。
提示:我们按照制造商标准方案进行 DNA 纯化。有关详细程序,请参阅此套件中的标准协议。


G. 精确定量和测序


1. 使用安捷伦高灵敏度 DNA 试剂盒按照制造方案精确定量 FACT-seq 文库的 DNA 浓度。
提示:有关详细程序,请参阅此套件中的协议。
2. 测序:在Illumina NovaSeq 6000测序仪或Illumina MiniSeq平台上对FACT-seq文库进行配对末端测序,部分结果(Zhao et al. , 2021)如图3所示。
提示:有关详细步骤,请参阅 Illumina 的协议。


 


图 3。 小鼠肾核 H3K27ac 测序文库的代表性基因组浏览器轨迹,来自Zhao等人。 (2021 年)。
结果来自小鼠 FFPE 肾核的 H3K27ac FACT-seq(- 表位检索)、小鼠 FFPE 肾核的 H3K27ac FACT-seq(+ 表位检索)、冷冻小鼠肾核的 H3K27ac CUT&Tag 、冷冻小鼠肾核的编码 H3K27ac ChIP - seq , 和CUT&Tag对小鼠肾细胞核的 IgG 对照。基因名称显示在底部。 Chr = 染色体。详细分析参考FACT-seq论文 (赵等,2021) 。




数据分析


1. 只有读取一个(R1)将参与下游分析。我们首先采用levenshtein距离算法将 T7 启动子序列映射到适配器修剪过程中的每个读取;然后可以使用内部脚本自定义脚本 ( https://github.com/pengweixing/FACT ) 从fastq文件中的每个读取中修剪 T7 启动子序列。
2. 参数 --end-to-end 的bowtie2 v.2.3.5 ( Langmead and Salzberg , 2012 )将修剪后的人类样本和小鼠样本的fastq文件分别映射到 hg19/hg38 参考基因组或 mm9/mm10 参考基因组--very-sensitive -I 10 -X 700。对齐的 BAM 文件由samtools v.1.9排序 (李等人,2009 年)。使用 Picard v1.79 ( http://picard.sourceforge.net ) 删除重复读数,并以 >q2 的对齐质量进行过滤。对于峰值调用,可以使用 SICER ( Zhang et al ., 2008 ) 调用宽峰,其参数间隙大小 = 600 bp 和窗口大小 = 200 bp。可以使用具有以下参数间隙大小 = 400 bp 和窗口大小 = 200 bp 的 SICER 调用窄峰。 bigwig 文件是使用deeptools ( Ramirez et al ., 2014 ) 从 BAM 文件生成的,参数为 bamCoverage - normalizeUsing CPM。测序文库的峰值通过 IGV ( ( Thorvaldsdottir 等人,2013 年)软件。


笔记


1. 使用脱氧胆酸钠、洋地黄皂苷和 SDS 等洗涤剂时要格外小心。将脱氧胆酸钠和洋地黄皂苷粉末溶解在液体中时,请记住戴上口罩、实验室外套和手套。
2. 我们已经成功地分析了 FFPE 小鼠细胞核中的 H3K27ac,输入量有限(1,000 个细胞核/管或从含有大约 5,000 个细胞核的一小块组织开始细胞核分离)。因此,即使核输入少于 100,000 个核/管,FACT-seq 协议通常也可以工作。
3. 表位检索条件是获得良好文库质量的重要步骤。在这里,我们提供了 H3K27ac、H3K27me3、H3K4me1 和 H3K36me3 的最佳表位检索条件。此表位检索条件可直接用于其他组蛋白修饰,包括 H3K4me3、H3K9me2 或 H3K9me3。
4. 如果您想测试未知条件,则针对 H3K27ac 的 FACT-seq 可能是阳性对照。
5. 我们在标记和纯化后得到的 DNA 量范围为 100 ng–300 ng/管(每管从 100,000 个 FFPE 核开始)。除非 DNA 浓度太低(< 10 ng/管)或 DNA 纯度太差(A 260/280 < 1.5),否则我们将始终进行文库制备。
6. 我们已经成功地在 FFPE 人结肠直肠癌和 FFPE 人胶质母细胞瘤样本中进行了 FACT-seq。使用不同的样本或分析不同的标记时,体外转录后的 RNA 产量会有所不同。如果特定样本的 RNA 产量较低,您可以尝试将体外转录的时间从过夜(8 小时)延长到 16 小时甚至更长。我们进行的最长体外转录大约是 23 小时,而且我们仍然有很好的文库。
7. 对于RNA纯化,我们最初使用传统的TRIzol RNA纯化,取得了不错的效果。由于TRIzol纯化耗时,我们最终改用试剂盒纯化。




食谱


1. 从 FFPE 组织切片中分离单核悬浮液的缓冲液


表 8. NST 缓冲液配方( N onidet P40 with S alts 和Tris缓冲液)
试剂 初始浓度 最终浓度 稀释倍数 最终体积 (10 毫升)
氯化钠 5,000 毫米 146 毫米 34.25 292微升
Tris-HCl pH 7.5 1,000 毫米 5毫米 200 50微升
Tris-HCl pH 8 1,000 毫米 5毫米 200 50微升
氯化钙2 1,000 毫米 1毫米 1000 10微升
氯化镁2 1,000 毫米 21 毫米 47.62 210微升
牛血清白蛋白 10% 0.05% 200 50微升
伊格帕尔 CA-630 10% 0.20% 50 200微升
无核酸酶水 / / / 9138微升


胶原酶:含 0.5 mM CaCl 2的 PBS 中 6 mg/mL
2的 PBS 中 600 单位/mL


2. 用于 Tn5 组装和活性测定的缓冲液








表 9 。 2 ×透析缓冲液配方


试剂 初始浓度 最终浓度 稀释倍数 最终体积 (10 毫升)
HEPES(K+) pH 7.2 500 毫米 100 毫米 5 2 毫升
氯化钠 5,000 毫米 200 毫米 25 400微升
乙二胺四乙酸 500 毫米 0.2 毫米 2500 4微升
数字地面电视 500 毫米 2毫米 250 40微升
海卫 X-100 10% 0.20% 50 200微升
甘油 100% 20% 5 2 毫升
无核酸酶水 / / / 5,356微升


表 10 。 Tn5组装配方


试剂 初始浓度 最终浓度 稀释倍数 最终体积 (50 μL )
Tn5MErev/Tn5ME-A 50微米 2微米 25 2微升
Tn5MErev/Tn5ME-B 50微米 2微米 25 2微升
甘油 100% 40% 2.5 20微升
2 ×透析缓冲液 / / / 15.24微升
Tn5 46.55微米 2微米 23.26 2.15微升
无核酸酶水 / / / 8.61微升


表 11 。 T7 pA-Tn5 组装配方


试剂 初始浓度 最终浓度 稀释倍数 最终体积 (50 μL )
Tn5MErev/Tn5ME-A 50微米 4微米 12.5 4微升
甘油 100% 40% 2.5 20微升
2 ×透析缓冲液 / / / 15.58微升
pA-Tn5 55.55微米 2微米 27.76 1.81微升
无核酸酶水 / / / 8.61微升


表 12. 2 × TD 缓冲液的配方


试剂 初始浓度 最终浓度 稀释倍数 最终体积 (10 毫升)
Tris-HCl pH 7.5 1,000 毫米 20毫米 50 200微升
氯化镁2 1,000 毫米 10毫米 100 100微升
二甲基甲酰胺 100% 20% 5 2 毫升
无核酸酶水 / / / 最多 10 毫升


3. 用于表位检索的缓冲液












表 13。 Epitope Retrieval Buffer-1的配方


试剂 初始浓度 最终浓度 稀释倍数 最终体积 (10 毫升)
乙二胺四乙酸 500 毫米 10毫米 50 200微升
Tris-HCl pH 8 1,000 毫米 50毫米 20 500微升
安全数据表 10% 0.10% 100 100微升
无核酸酶水 / / / 9.2 毫升




表 14. Epitope Retrieval Buffer-2 的配方


试剂 初始浓度 最终浓度 稀释倍数 最终体积 (10 毫升)
乙二胺四乙酸 500 毫米 10毫米 50 200微升
Tris-HCl pH 8 1,000 毫米 50毫米 20 500微升
安全数据表 10% 0.10% 100 100微升
脱氧胆酸钠 10% 0.10% 100 100微升
无核酸酶
水 / / / 9.1 毫升


4. 用于抗体结合和标记的缓冲液


表 15. FACT-seq Dig-washing 缓冲液的配方(用于 8 个文库)


试剂 初始浓度 最终浓度 稀释倍数 最终体积 (15 毫升)
HEPES(K+) pH 7.6 500 毫米 20毫米 25 600微升
氯化钠 5,000 毫米 150 毫米 33.33 450微升
亚精胺 2,000 毫米 0.5 毫米 4,000 3.75微升
洋地黄皂苷 5% 0.05% 100 150微升
伊格帕尔 CA-630 10% 0.01% 1,000 15微升
牛血清白蛋白 10% 1% 10 1,500微升
无核酸酶水 
(添加蛋白酶抑制剂) / / 12.28 毫升


要制备 FACT-seq 抗体缓冲液(用于 8 个文库),将 4 mL的 FACT-seq Dig-washing 缓冲液转移到新试管中,加入 16 μL的 0.5 M EDTA,充分混合并保持在冰上。


表 16。 FACT-seq Dig-300 缓冲液配方(适用于 8 个文库)


试剂 初始浓度 最终浓度 稀释倍数 最终体积 (10 毫升)
HEPES(K+) pH 7.6 500 毫米 20毫米 25 400微升
氯化钠 5,000 毫米 300 毫米 16.67 600微升
亚精胺 2,000 毫米 0.5 毫米 4,000 2.5微升
洋地黄皂苷 5% 0.05% 100 100微升
伊格帕尔 CA-630 10% 0.01% 1,000 100微升
牛血清白蛋白 10% 1% 10 1,000微升
无核酸酶水 
(添加蛋白酶抑制剂) / / 7.89 毫升


表 17。 FACT-seq 标记缓冲液的配方(用于 8 个库)


试剂 初始浓度 最终浓度 稀释倍数 最终体积 (2 毫升)
HEPES (K+) pH 7.6 500 毫米 20毫米 25 80微升
氯化钠 5,000 毫米 300 毫米 16.67 120微升
亚精胺 2,000 毫米 0.5 毫米 4,000 0.5微升
洋地黄皂苷 5% 0.05% 100 20微升
伊格帕尔 CA-630 10% 0.01% 1000 2微升
氯化镁2 1,000 毫米 10毫米 100 20微升
无核酸酶水 
(添加蛋白酶抑制剂) / / 1,757.5微升


5. 用于文库制备的缓冲液


表 18.挤压浸泡缓冲液配方


试剂 初始浓度 最终浓度 稀释倍数 最终体积 (10 毫升)
氯化钠 5,000 毫米 500 毫米 10 1,000微升
乙二胺四乙酸 500 毫米 1毫米 500 20微升
安全数据表 10% 0.50% 20 500微升
无核酸酶水 / / / 8,480微升




致谢


该协议源自 Chen Lab (Zhao et al. , 2021) 之前的出版物 FACT-seq。我们要感谢参与这项工作的所有陈实验室成员的贡献。我们要感谢卡罗林斯卡研究所的蛋白质科学设施提供纯化的 Tn5 和 pA-Tn5。我们的资金来源包括:瑞典研究委员会 [VR-2016-06794, VR-201702074 to XC];奥克 维伯格斯 僵硬[M20-0007 至 XC ];北尔基金会(至 XC);杰森基金会(至 XC); Petrus och Augusta Hedlunds Stiftelse (至 XC); Göran Gustafsson 的年轻研究人员奖(授予 XC); Vleugel基金会(至 XC); Linnéstiftelsen for medicinsk 分叉(到 XC);乌普萨拉大学(至 XC);瑞典癌症协会 (CAN 2021-1449Pj, 22 0491 JIA to XC)。


利益争夺


Xingqi Chen 、Vamsi Krishna Polavarapu和Linxuan Zhao已提交与先前发表的 FACT-seq 论文和此处描述的协议相关的专利申请。专利申请的标题是 “从福尔马林固定石蜡包埋 (FFPE) 组织样本中制备 DNA 的方法”。瑞典临时 申请于 2021 年 6 月 28 日在瑞典提交,专利申请号2150823-9 。作者声明没有竞争的经济利益。


参考


Astolfi, A., Urbini, M., Indio, V., Nannini, M., Genovese, CG, Santini, D., Saponara, M., Mandrioli, A., Ercolani, G., Brandi, G.等人_ (2015 年)。胃肠道间质瘤 (GIST) 中福尔马林固定、石蜡包埋 (FFPE) 肿瘤组织的全外显子组测序 (WES)。 BMC 基因组学16:892。
Becker, JS, McCarthy, RL, Sidoli, S., Donahue, G., Kaeding, KE, He, Z., Lin, S., Garcia, BA 和 Zaret, KS (2017)。异染色质的基因组和蛋白质组学分辨率及其对替代命运基因的限制。 摩尔细胞68(6):1023-1037 e1015。
Bolognesi, C., Forcato, C., Buson, G., Fontana, F., Mangano, C., Doffini, A., Sero, V., Lanzellotto, R., Signorini, G., Calanca, A.,等人。 (2016 年)。纯细胞群的数字分选能够通过下一代测序对异质福尔马林固定石蜡包埋肿瘤进行明确的遗传分析。 科学代表6:20944。
Buenrostro, JD, Wu, B., Litzenburger, UM, Ruff, D., Gonzales, ML, Snyder, MP, Chang, HY 和 Greenleaf, WJ (2015)。单细胞染色质可及性揭示了调控变异的原理。 自然523(7561):486-490。
Cejas, P., Li, L., O'Neill, NK, Duarte, M., Rao, P., Bowden, M., Zhou, CW, Mendiola, M., Burgos, E., Feliu, J.,等人。 (2016 年)。来自固定临床组织的染色质免疫沉淀揭示了肿瘤特异性增强子谱。 Nat Med 22(6):685-691。
Fanelli, M.、Amatori, S.、Barozzi, I. 和 Minucci, S. (2011)。来自石蜡包埋的病理组织的染色质免疫沉淀和高通量测序。 国家协议6(12):1905-1919。
Fanelli,M.,Amatori,S.,Barozzi,I.,Soncini,M.,Dal Zuffo,R.,Bucci,G.,Capra,M.,Quarto,M.,Dellino,GI,Mercurio,C.,等人。 (2010)。病理组织染色质免疫沉淀,加上高通量测序,可以对患者样本进行表观遗传分析。 Proc Natl Acad Sci USA 107(50):21535-21540。
Font-Tello, A., Kesten, N., Xie, Y., Taing, L., Vareslija, D., Young, LS, Hamid, AA, Van Allen, EM, Sweeney, CJ, Gjini, E., et人_ (2020 年)。 FiTAc-seq:用于 H3K27ac 分析和 FFPE 组织的超增强子分析的固定组织 ChIP-seq。 国家协议15(8):2503-2518 。
Fox, CH, Johnson, F..B, Whiting, J. 和 Roller, PP (1985)。甲醛固定。 J Histochem Cytochem 33(8):845-53。
Haile,S.,Corbett,RD,Bilobram,S.,Bye,MH,Kirk,H.,Pandoh,P.,Trinh,E.,MacLeod,T.,McDonald,H.,Bala,M.,等. (2019)。福尔马林固定石蜡包埋样品基因组 DNA 的下一代测序中错误序列和伪影嵌合读数的来源。 核酸研究47(2):e12。
Kaya-Okur, HS, Wu, SJ, Codomo, CA, Pledger, ES, Bryson, TD, Henikoff, JG, Ahmad, K. 和 Henikoff, S. (2019)。 CUT&Tag 用于小样本和单细胞的有效表观基因组分析。 国家公社10(1):1930。
Langmead, B. 和 Salzberg, SL (2012)。使用 Bowtie 2 进行快速间隙读取对齐。 Nat 方法9(4):357-359。
Li, H.、Handsaker, B.、Wysoker, A.、Fennell, T.、Ruan, J.、Homer, N.、Marth, G.、Abecasis, G.、Durbin, R. 和 Genome Project Data Processing, S. (2009)。序列比对/映射格式和 SAMtools。 生物信息学25(16):2078-2079。
Pennock, ND, Jindal, S., Horton, W., Sun, D., Narasimhan, J., Carbone, L., Fei, SS, Searles, R., Harrington, CA, Burchard, J.等。 (2019)。来自档案 FFPE 乳腺癌样本的 RNA-seq:分子通路保真度和新发现。 BMC Med 基因组学12(1):195。
Picelli, S.、Bjorklund, AK、Reinius, B.、Sagasser, S.、Winberg, G. 和 Sandberg, R. (2014)。用于大规模测序项目的 Tn5 转座酶和标记程序。 基因组研究24(12):2033-2040。
Ramirez, F.、Dundar, F.、Diehl, S.、Gruning, BA 和 Manke, T. (2014)。 deepTools:探索深度测序数据的灵活平台。 Nucleic Acids Res 42(网络服务器问题):W187-191。
Teytelman, L.、Ozaydin, B.、Zill, O.、Lefrancois, P.、Snyder, M.、Rine, J. 和 Eisen, MB (2009)。染色质结构对基因组分析 DNA 处理的影响。 公共科学图书馆一号4(8):e6700。
Thorvaldsdottir, H.、Robinson, JT 和 Mesirov, JP (2013)。 Integrative Genomics Viewer (IGV):高性能基因组数据可视化和探索。 简要 Bioinform 14(2):178-192。
Waldron, L.、Simpson, P.、Parmigiani, G. 和 Huttenhower, C. (2012)。转化生物信息学新兴技术报告:档案组织基因表达谱研讨会。 BMC 癌症12:124。
Wang, Y.、Moorhead, M.、Karlin-Neumann, G.、Falkowski, M.、Chen, C.、Siddiqui, F.、Davis, RW、Willis, TD 和 Faham, M. (2005)。使用分子倒置探针 (MIP) 进行等位基因定量。核酸研究 33(21):e183。
Zhang, Y., Liu, T., Meyer, CA, Eeckhoute, J., Johnson, DS, Bernstein, BE, Nusbaum, C., Myers, RM, Brown, M., Li, W., et al 。 (2008 年)。 ChIP-Seq (MACS) 的基于模型的分析。 基因组生物学9(9):R137。
Zhao, L., Xing, P., Polavarapu, VK, Zhao, M., Valero-Martinez, B., Dang, Y., Maturi, N., Mathot, L., Neves, I., Yildirim, I.等。 _ (2021 年)。 FACT-seq:分析福尔马林固定石蜡包埋样品中细胞数较少的组蛋白修饰。 核酸研究49(21):e125。




登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2022 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zhao, L., Polavarapu, V. K., Yadav, R. P., Xing, P. and Chen, X. (2022). A Highly Sensitive Method to Efficiently Profile the Histone Modifications of FFPE Samples. Bio-protocol 12(10): e4418. DOI: 10.21769/BioProtoc.4418.
提问与回复

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。