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Isolation of Nuclei in Tagged Cell Types (INTACT), RNA Extraction and Ribosomal RNA Degradation to Prepare Material for RNA-Seq
从标记的细胞类型中分离细胞核、提取RNA并去除核糖体RNA以用于RNA-Seq 分析   

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本实验方案简略版
Plant Physiology
Feb 2018

Abstract

Gene expression is dynamically regulated on many levels, including chromatin accessibility and transcription. In order to study these nuclear regulatory events, we describe our method to purify nuclei with Isolation of Nuclei in TAgged Cell Types (INTACT). As nuclear RNA is low in polyadenylated transcripts and conventional pulldown methods would not capture non-polyadenylated pre-mRNA, we also present our method to remove ribosomal RNA from the total nuclear RNA in preparation for nuclear RNA-Seq.

Keywords: Gene expression (基因表达), Nuclear purification (核纯化), INTACT (INTACT), rRNA degradation (rRNA降解), RNA extraction (RNA提取), RNA-Seq (RNA-Seq)

Background

Isolating specific cell types for gene expression experiments reduces the noise and increases the precision of the experiment and the number of differently expressed genes found. Various methods for cell type-specific studies are widely used, each one with their strengths and weaknesses (reviewed in Bailey-Serres, 2013). Isolating specific regulatory compartments, such as nuclei (from organelles, ribosomes, cytosol, etc.) provides further precision in dissecting the molecular events in regulation of gene expression. Here we describe a method that allows isolating nuclei of specific cell types from frozen tissue, suited for experiments where nuclear gene expression is to be studied (e.g., RNA-Seq of nuclear RNA, ATAC-Seq, ChIP-Seq, etc.). Furthermore, we describe the processing of RNA that leads to material suited to be the input for an RNA-Seq experiment. The protocols described here were used with rice root tissue (Oryza sativa cv. Nipponbare) (Reynoso et al., 2018), but they are based on previous protocols developed for Arabidopsis (Deal and Henikoff, 2010 and 2011) and tomato (Ron et al., 2014).

The first part of this protocol, the INTACT method (for Isolation of Nuclei Tagged in specific Cell Types) allows in vivo affinity labeling and subsequent purification of nuclei from a cell type of interest. This is achieved through cell type-specific expression of a tripartite nuclear tagging fusion protein (NTF) consisting of a nuclear envelope targeting domain, GFP, and the biotin ligase recognition peptide (BLRP). Co-expression of NTF along with the E. coli biotin ligase gene codon optimized for rice, BirA, in the cell type of interest results in the production of fluorescently labeled, biotinylated nuclei specifically in that cell type. These labeled nuclei can then be affinity purified from a crude tissue homogenate using streptavidin-coated magnetic beads, thus allowing access to RNA and chromatin from the cell type of interest.

The second part of the method describes the processing of nuclear RNA to produce a sample that is suitable to be the input for RNA-Seq library preparation. Typically for eukaryotic samples, ribosomal RNA (rRNA) and organellar RNA is removed from RNA samples by polyA-pulldown methods. However, nuclear mRNA contains pre-mRNA in many stages of processing, most not polyadenylated, while many of the polyadenylated mRNAs are rapidly exported. Hence, to profile the transcripts at different stages of processing, polyA-isolation methods are not suited. Inspired by previous work (Morlan et al., 2012; Gregory Smaldone, personal communication), we developed a method to remove ribosomal RNA (rRNA) from rice samples, specifically using the INTACT-purified nuclei as our input. The steps of this method include isolation of total RNA from INTACT-purified nuclei, removal of residual genomic DNA, annealing of DNA probes to rRNA, degradation of rRNA with RNase specific to RNA:DNA hybrids, and degradation of the DNA probes. At the end of the protocol, the RNA sample is ready for RNA-Seq library construction.


Part I: INTACT purification of nuclei

Materials and Reagents

  1. Pipette tips (P20, P200 and P1000, nuclease-free, e.g., USA Scientific, catalog numbers: 1123-1710 , 1120-8780 , 1126-7510 )
  2. (Optional) 13 ml Falcon tube
  3. 30 μm cell strainer (Sysmex, CellTrics, catalog number: 04-004-2326 )
  4. 15 ml Falcon tubes (nuclease-free, e.g., VWR, catalog number: 89039-666 )
  5. 1.5 ml Eppendorf tubes (nuclease-free, e.g., Denville Scientific, catalog number: C2170 )
  6. Pasteur pipettes (nuclease-free, e.g., Phenix Research Products, catalog number: PP-137038C )
  7. Slide 
  8. Coverslip 
  9. PCR tube strips (nuclease-free, e.g., USA Scientific, catalog number: 1402-2708
  10. 0.22 μm syringe filters (e.g., Merck, catalog number: SLGP033RB )
  11. 50 ml Falcon tubes (nuclease-free, e.g., VWR, catalog number: 89039-658 )
  12. Aluminum foil
  13. Transgenic plant tissue with biotin-tagged nuclei
  14. INTACT binary vectors (Ron et al., 2014; Reynoso et al., 2018)
    Note: They will be available at https://gateway.psb.ugent.be/search. These plasmids allow inserting a T-DNA with the promoter of your choice into the plant species of your choice.
  15. Liquid nitrogen
  16. M-280 Streptavidin Dynabeads (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11205D )
  17. NaOH
  18. MOPS (Sigma-Aldrich, catalog number: M1254-25G )
  19. Sodium chloride (NaCl) (e.g., Fisher Scientific, catalog number: S271 )
  20. Potassium chloride (KCl) (e.g., Fisher Scientific, catalog number: BP366-1 )
  21. Ethylenediaminetetraacetic acid (EDTA) (e.g., Fisher Scientific, catalog number: S311 )
  22. EGTA (Sigma-Aldrich, catalog number: E3889-25G )
  23. Spermine (Sigma-Aldrich, catalog number: S1141 )
  24. Spermidine (Sigma-Aldrich, catalog number: S2626 )
  25. Protease Inhibitor Cocktail (Sigma-Aldrich, catalog number: P9599 ) or cOmplete mini protease inhibitor tablets EDTA-free (Roche Diagnostics, catalog number: 11836170001 )
  26. Triton X-100 (Sigma-Aldrich, catalog number: 648466 )
  27. Propidium Iodide (PI) stain (Sigma-Aldrich, catalog number: P4170 )
  28. 10 N NaOH (see Recipes)
  29. 0.5 M MOPS, pH 7 (see Recipes)
  30. 1 M NaCl (see Recipes)
  31. 2 M KCl (see Recipes)
  32. 0.5 M EDTA (see Recipes)
  33. 0.5 M EGTA (see Recipes)
  34. 0.2 M spermine (see Recipes)
  35. 0.2 M spermidine (see Recipes)
  36. 10% Triton X-100 (see Recipes)
  37. Nuclei purification buffer base (see Recipes)
  38. NPB and NPBt buffers (see Recipes)
  39. Propidium Iodide (PI) stains (see Recipes)

Equipment

  1. Pipettes (P20, P200, P1000; e.g., Eppendorf, catalog numbers: 3123000039 , 3123000055 , 3123000063 )
  2. Ceramic mortars and pestles (wipe clean with RNaseZap [Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9780 ] or other RNase removal product)
  3. Centrifuge for 15 ml Falcon tubes (e.g., Eppendorf, model: 5810 R )
  4. DynamagTM-15 Magnet (Thermo Fisher Scientific, model: DynamagTM-15, catalog number: 12301D )
    Note: Can be replaced with a homemade version. For example, a strong rare earth (neodymium) magnet (e.g., 1 x 10 cm bar) can be taped inside a 50 ml Falcon tube and padding can be added to keep 15 ml Falcon tubes in place next to the magnet (see Figure 1).


    Figure 1. Homemade magnet for two 15 ml Falcon tubes. Two neodymium magnets are placed inside 50 ml Falcon tubes held in a tube rack. The attraction between the two magnets holds them in place. To prop the 15 ml tubes to correct position against the magnet, paper towel is taped inside the 50 ml tube.

  5. DynamagTM-2 Magnet (Thermo Fisher Scientific, model: DynamagTM-2, catalog number: 12321D ), or a homemade version of this
  6. Hemocytometer (SCK Films, In-Cyto, catalog number: DHC-N01-2 )
  7. BD AdamsTM NutatorTM Single-Speed Orbital Mixer (e.g., Fisher Scientific, catalog number: 14-062)
    Manufacturer: BD, catalog number: 421105/DEL .
  8. Fridge and/or cold room at 4 °C
  9. Fluorescent microscope with filters to visualize either DAPI or PI stain (Edmund Optics, catalog numbers: 86-371 , 67-008 )

Procedure

Figure 2 provides the overview of the procedure, including optional check points.


Figure 2. Flowchart summarizing the steps in Part I: INTACT purification of nuclei

Note: For researchers who plan to use INTACT-purified nuclei for Chromatin ImmunoPrecipitation (ChIP) experiments (not described in this protocol): to carry out ChIP experiments, fresh tissue needs to be cross-linked.

  1. Crosslink proteins to DNA by placing all starting tissue in 30 ml of NPB with 1% formaldehyde (Sigma-Aldrich , F8775) in a 50-ml Falcon tube, and incubating the tissue under vacuum for 10 min.
  2. Add glycine (Fisher Scientific, BP381) to a final concentration of 0.125 M, place under vacuum for 5 more min.
  3. Wash tissue 3 times with water and blot dry. 

  1. Isolating nuclei from frozen material
    1. Prepare fresh NPB and NPBt buffers as needed (~15 ml NPB and 45 ml NBPt per sample) and cool the buffers on ice. We recommend processing maximum eight samples in one go.
    2. Grind frozen tissue (50-200 1 cm rice root tips. 200 root tips weigh 180 mg) in liquid nitrogen with ceramic mortars and pestles until very fine.
      Optional: Pour powder into 13 ml Falcon tubes and weigh the sample. Cool everything with liquid nitrogen and do not let the samples thaw.
    3. Add ~8 ml of ice-cold NPB with a transfer pipette into the mortar and resuspend the powdered tissue by gently grinding it (scale up for larger samples). It is important that NPB does not freeze, so either use minimal amounts of liquid nitrogen for grinding or move to a fresh mortar for this step.
    4. Filter the resuspension through the 30 μm cell strainer into a 15 ml Falcon tube, and place the Falcon tube on ice while you process rest of your samples.
    5. Centrifuge the filtered nuclei 1,000 x g for 15 min at 4 °C (2,229 rpm on the Eppendorf 5810 R centrifuge).
    6. Prepare the streptavidin beads in 1.5 ml tubes (one per sample). Wash 25 μl of Invitrogen M-280 Streptavidin Dynabeads for each sample. To wash the beads, pipette well-mixed beads into a 1.5 ml tube, place on a magnet, remove supernatant, add and mix with 1 ml NPB, place on the magnet, remove supernatant, resuspend in NPB of the original volume (50 μl).
    7. Move the Falcon tubes from the centrifuge onto ice.
      Optional: Mark down the volume of supernatant (SN) and set aside 10% of the supernatant for a Western blot (‘SN1000’–if you want to confirm you have not lost any nuclei/biotin signal in your supernatant).
      1. Discard the supernatant (by careful pouring).
      2. Resuspend the nuclei in 1 ml of cold NPB by pipetting up and down. In other words, releasing buffer from the pipette on top of the nuclear pellet until the pellet fully resuspends.
      3. Set aside 11 μl of the resuspended nuclei for quantification and yield calculations (PCR strip works well). See Procedure B.
        Optional: Set aside 10% of the resuspension ‘P1000’ (Pellet) for a Western blot (the total amount of biotin signal in the sample).
    8. Transfer the resuspended nuclei (~1 ml) onto the washed beads (in 1.5 ml tubes). Rotate the tubes on a nutator (at ~20 rpm) at 4 °C for 30 min to bind nuclear envelope biotin onto streptavidin on the beads.
    9. Dilute the 1 ml of bead-nuclei mixture with 14 ml NPBt in a 15 ml Falcon tube. Mix gently and place on the nutator (at ~20 rpm) at 4 °C for 30 sec. Place the tube on the magnet at 4 °C for 15-20 min.
      Optional: Set aside 10% of the supernatant for a Western blot (‘Unbound’).
    10. Carefully remove the supernatant with a Pasteur pipette and gently resuspend the beads in 14 ml NPBt. Mix gently and place on the nutator (at ~20 rpm) at 4 °C for 30 sec. Place the tube on the magnet for 10 min (at 4 °C).
    11. Repeat Step A10.
    12. Gently remove the supernatant. Resuspend the beads in 1 ml of NPBt.
      Set aside 11 μl of the nuclei for quantification and yield calculations (PCR strip works well). See Procedure B.
      Optional: Set aside 10% of the resuspended beads for a Western blot (‘Bound’ to make sure most of your biotinylated signal is here).
    13. Transfer 1 ml resuspension into a 1.5 ml tube and capture on the magnet.
    14. Remove supernatant, resuspend beads in 20 μl NPB.
      Note: Do not freeze nuclei until after they have been counted and aliquoted.

  2. Counting nuclei with a hemocytometer to quantify the yield
    1. Mix 11 μl nuclei (either on beads from Step A14 or from the resuspended pellet on Step A7c) with 0.5 μl 10 μg/ml Propidium Iodide (PI) stain. Allow nuclei to stain for at least 5 min before imaging.
    2. Pipette 10 μl of the sample onto a hemocytometer. One hemocytometer has two positions, A and B, for counting nuclei so you need one hemocytometer for counting two samples. The sample is pipetted onto a half-moon shaped area, which pushes the nuclei resuspension between the slide and the coverslip (Figure 3).
    3. View the samples under fluorescent light and a filter suitable for PI, count the nuclei using the counting grid (Figure 3). Use this to calculate the number of nuclei in total sample. 


      Figure 3. Appearance of the counting grid on a hemacytometer, and visualization of PI-stained nuclei with confocal laser scanning microscopy

      1. For example, you can calculate 16 squares in the corner of the grid, comprising 1/9th of the whole grid (this area is 1 x 1 x 0.1 mm, or 0.1 μl, and represents 1/10,000th of 1 ml).
      2. Multiply the number of nuclei found inside this area by 10,000 to find out total yield (how many nuclei you have isolated).
        Advice:
        1. Typical yields for a strong near-constitutive promoter line (e.g., 35Spro:NTF) from 200 root tips is 150,000-230,000 nuclei.
        2. Check/count starting number of total nuclei (using nuclei from Step A7c) if you have concerns about your original nuclear isolation and appearance of the nuclei under the microscope.
      3. Optional calculations: Background can be calculated by using non-transgenic tissue (‘negative control’) and comparing to constitutive promoter (‘35S-IN’), using both nuclei pulled down with beads and number of total nuclei in the original pellet (P1000):
        Efficiency: (35S-IN beads - background)/(35S-IN pellet)
        Background: (35S-IN beads) x (negative control beads)/(negative control pellet) 

  3. Aliquoting nuclei for RNA and ATAC.
    Note: This splitting of the sample is relevant only if you are using the isolated nuclei for an ATAC-seq experiment (described in detail by Bajic et al., 2018) in addition to RNA-Seq.
    1. Calculate how many μl of nuclei you need out of the final 20 μl to get 50,000 nuclei for ATAC. Pipette these onto a PCR strip on ice and start ATAC protocol without freezing (Bajic et al., 2018).
    2. Use the rest of the nuclei for RNA immediately, or freeze at -80 °C for later RNA work.

Data analysis

The protocol described above needs to be combined with other downstream protocols (RNA-Seq, ATAC-Seq, ChIP-Se q, qRT-PCT) to generate data to be analyzed.

Recipes

  1. 10 N NaOH
    To 80 ml of dH2O, slowly add 40 g of NaOH pellets while continuously stirring
  2. 0.5 M MOPS pH 7
    20.93 g MOPS in 200 ml deionized water
    Adjust the pH to 7.0 with 10 N NaOH (or NaOH pellets)
    Filter sterilize through 0.22 μm filter, aliquot to 40 ml aliquots in 50 ml Falcon tubes, and store at -20 °C
  3. 1 M NaCl
    11.688 g NaCl in 200 ml deionized water
    Autoclave, store at room temperature
  4. 2 M KCl
    29.8205 g KCl in 200 ml deionized water
    Autoclave, store at room temperature
  5. 0.5 M EDTA
    29.224 g EDTA in 200 ml deionized water
    Adjust pH to 8.0 with solid NaOH (> 8 g)
    Note: EDTA does not dissolve until the pH has been adjusted.
    Autoclave, store at room temperature
  6. 0.5 M EGTA
    19.0175 g EGTA in 100 ml deionized water
    Adjust pH 8.0 with solid NaOH (~4 g)
    Note: EGTA does not dissolve until the pH has been adjusted.
    Autoclave, store at room temperature
  7. 0.2 M spermine
    1 g in 24.7 ml of deionized water
    Filter sterilize through 0.22 μm filter, aliquot, and freeze at -20 °C
  8. 0.2 M spermidine
    1 g in 34.4 ml of deionized water.
    Filter sterilize through a 0.22 μm filter, aliquot, and freeze at -20 °C
  9. 10% Triton X-100
    5 ml Triton X-100
    45 ml nuclease-free water
    Keep at room temperature
  10. Nuclei Purification Buffer (NPB) base
    NPB base can be filter sterilized through a 0.22 μm filter and kept at 4 °C for up to 3 months. Typically 60 ml of NPB base is needed per sample
    20 mM MOPS (pH 7)
    40 mM NaCl
    90 mM KCl
    2 mM EDTA
    0.5 mM EGTA
    For 60 ml (one sample):
    2.4 ml 0.5 M MOPS
    2.4 ml 1 M NaCl
    2.7 ml 2 M KCl
    240 μl 0.5 M EDTA pH 8
    60 μl 0.5 M EGTA pH 8
    For 1 L (~16 samples):
    40 ml 0.5 M MOPS
    8 ml 5 M NaCl
    45 ml 2 M KCl
    4 ml 0.5 M EDTA pH 8
    1 ml 0.5 M EGTA pH 8
  11. NPB and NPBt buffers

    Add Triton X-100, spermidine, spermine and cOmplete protease inhibitors just prior to use and keep on ice or at 4 °C
  12. Propidium Iodide (PI) stains
    Prepare 1,000x concentrated stock solution by dissolving 10 mg PI in 1 ml distilled water
    Prepare a 1x working solution of 10 μg/ml of PI with diluting the stock 1:1,000 with distilled water
    Store solutions at 4 °C, wrapped in aluminum foil to prevent degradation

Part II: Total RNA isolation and rRNA degradation

Materials and Reagents

  1. Pipette tips (for P10, P20, P200, P1000, nuclease-free e.g., USA Scientific, catalog numbers: 1123-1710 , 1120-8780 , 1126-7510 )
  2. 1.5 ml Eppendorf tubes (nuclease-free, e.g., Denville Scientific, catalog number: C2170 )
  3. RNase-free PCR strips (e.g., USA Scientific, catalog number: 1402-2708 )
  4. 0.22 μm syringe filters (e.g., Merck, catalog number: SLGP033RB )
  5. Nuclei isolated with INTACT in Part I, or any other suitable starting tissue
  6. 1 μM stock of DNA probes designed against rRNA sequences
    Note: See detailed description on how to design these in Step 1 of the procedure.
  7. RNeasy Micro Kit (QIAGEN, catalog number: 74004 ) or RNeasy Micro Plus Kit (QIAGEN, catalog number: 74034 )
    Note: Both kits contain the needed components, but often one is available cheaper than the other.
  8. 70% ethanol
  9. RNase-free water
  10. Turbo DNase Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM2238 )
  11. Agencourt RNAClean XP beads (Beckman Coulter, catalog number: A66514 )
    Alternatively: Use AMPureXP beads (Beckman Coulter, catalog number: A63881 ).
  12. Hybridase Thermostable RNase H (Epicentre, catalog number: H39500 )
  13. Tris base (e.g., Fisher Scientific, catalog number: BP154-1 )
  14. Hydrochloric acid (HCl, e.g., Sigma-Aldrich, catalog number: 435570 )
  15. Sodium chloride (NaCl) (e.g., Fisher Scientific, catalog number: S271 )
  16. Magnesium chloride (MgCl2) (e.g., Sigma-Aldrich, catalog number: M8266 )
  17. 5x Hybridization buffer H1 (see Recipes)
  18. 10x Hybridase buffer H2 (see Recipes)

Equipment

  1. Pipettes (P20, P200, P1000; e.g., Eppendorf, catalog numbers: 3123000039 , 3123000055 , 3123000063 )
  2. DynamagTM-2 Magnet (Thermo Fisher Scientific, model: DynamagTM-2, catalog number: 12321D ), or a homemade version of this
  3. Magnetic rack for PCR strips (e.g., Edge BioSystems, catalog number: 57624 )
  4. Microcentrifuge (e.g., Eppendorf, catalog number: 5424 series )
  5. PCR machine (e.g., Thermo Fisher Scientific, Applied BiosystemsTM, model: 2720 )
  6. NanoDrop spectrophotometer (e.g., Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000 , catalog number: ND-2000)

Software

  1. Sequence analysis software: SnapGene, Geneious, etc.

Procedure

Figure 4 provides the overview of the protocol, including all the possible stop points and temperatures (theoretically you can stop at these points for up to multiple weeks).


Figure 4. Flowchart summarizing the steps in Part II: Total RNA isolation and rRNA degradation

  1. Designing DNA probes against rRNA sequences
    1. Designing the DNA probes against rRNA sequences is a key part of this procedure, as this is how you target RNA for degradation. For targeting nuclear rRNA, you need to obtain the sequences of 5S, 5.8S, 18S and 25S rRNAs and their internal and external transcribed spacers. If you plan to carry out rRNA degradation for total cellular RNA, remember to also target mitochondrial and plastid rRNAs. For many species, these sequences can be found from https://www.ncbi.nlm.nih.gov/nucleotide/.
    2. Design your probes to be 60 bp long, cover the entire length of rRNA (including the spacer regions as they will be present in nuclear rRNA), and to reverse complement the rRNA sequences. Any sequence analysis software able to convert sequences to reverse complement (SnapGene, Geneious, etc.) can be used.
    3. Order your probes (DNA oligos) as a pre-made mix at 10 nmol in 1,000 μl of water. This is your 10 μM stock.
    4. Dilute your stock 10x into a working mix where each probe is at 1 μM.
  2. Total RNA isolation with QIAGEN RNeasy Micro Kit from nuclei isolated using INTACT
    Purify the nuclear RNA using the QIAGEN RNeasy Micro Kit as follows:
    1. Start by adding 350 μl of lysis buffer RLT (with 10 μl beta-mercaptoethanol/1 ml added) to the 20 μl of purified nuclei. Vortex vigorously for 2 min.
    2. Centrifuge the lysate at 1,000 x g (3,100 rpm) for 2 min at RT to pellet the beads. Place the tube onto a DynamagTM-2 Magnet and transfer the supernatant to a new 1.5 ml tube. Add 350 μl of 70% ethanol and vortex several times to mix.
    3. Pipette lysate/ethanol mixture into an RNeasy MinElute spin column resting in a 2 ml collection tube and centrifuge at 10,000 x g (9,700 rpm) for 1 min at RT. Discard flowthrough.
    4. Add 350 μl of buffer RW1 to the column. Centrifuge at 10,000 x g (9,700 rpm) for 1 min at RT. Discard flowthrough and move the column to a new 2 ml collection tube.
    5. Add 500 μl of buffer RPE to the column. Centrifuge at 10,000 x g (9,700 rpm) for 1 min at RT. Discard flowthrough.
    6. Add 500 μl of 80% ethanol to the column. Centrifuge at 10,000 x g (9,700 rpm) for 1 min at RT. Discard flowthrough and move the column to a new 2 ml collection tube.
    7. Open the column lid and centrifuge at top speed (16,000 x g) for 5 min at RT. Discard the flowthrough and place the column into a new 1.5 ml tube.
    8. Add 20 μl RNase-free water onto the column membrane and allow to stand for 1 min. Centrifuge at 16,000 x g for 1 min at RT.
    9. Store RNA at -80 °C. 
  3. DNase I treatment to eliminate genomic DNA contamination
    Use DNase I protocol for Turbo DNase I as follows:
    1. Add to 20 μl of RNA:
      2 μl of 10x DNase I reaction buffer
      1 μl DNase I
    2. Incubate for 30 min at 37 °C.
    3. Add 2 μl DNase I inactivation reagent (vortex well before adding) and incubate for 5 min at RT (vortex every 1 min).
    4. Spin down (2,000 x g for 5 min) and recover 20 μl into a new tube.
  4. RNA cleanup using Agencourt RNAClean XP bead to remove DNase buffer
    1. Add 1.8 volume (e.g., 36 μl for 20 μl of RNA) of RNAClean XP beads to the reaction.
    2. Incubate at RT for 10 min.
    3. Onto a magnet for 5 min.
    4. Remove most of the supernatant (leave 5 μl behind to avoid pipetting up beads).
    5. Leave tubes on the magnet, add 200 μl 70% ethanol, let stand for 30 sec.
    6. Remove all the supernatant.
    7. Repeat 70% wash as above once more (two washes total).
    8. Remove as much of the EtOH as possible.
    9. Air dry beads on the magnet for 10 min (until appear dry, see Figure 5).
    10. Add 15 μl RNase-free water to the first aliquot dried bead and mix.
    11. If you have aliquoted samples, pool back together.
    12. Incubate at RT for 5 min.
    13. Onto the magnet for 5 min.
    14. Recover 15 μl eluate.


      Figure 5. Appearance of Agencourt XP beads. A. After ethanol has been removed, the beads appear glistening and wet. B. Beads ready for elution look dry, the pellet is thinner and often lighter in color.

  5. NanoDrop for [RNA] and 260/280 so that you know the RNA concentration for adjusting probe concentration.
  6. rRNA probe hybridization to bind DNA probes to rRNA
    1. Reaction size for Steps 6 and 7 needs to be 6 μl + 4 μl (total 10 μl) for the buffers to be optimal for each step. You can prepare multiple aliquots of the reaction and pool back before DNase I treatment (Step 8). Successful RNA-Seq libraries have been made from a single 0.1 μg reaction.
    2. Add following together:
      3.8 μl RNA (if this would be more than 1 μg RNA, make up with RNase-free water)
      1.2 μl 5x Hybridization buffer H1 (Recipe 1)
      1.0 μl probe mix–choose your concentration as follows:
      1. If your RNA amount is 1 μg, use 1 µM/oligo working stock of probe mix.
      2. If your RNA amount is 0.1 μg, dilute probes to 0.1 µM/oligo.
        Note: This is the most common probe concentration in our experiments.
      3. If your RNA amount is 0.01 μg, dilute probes to 0.01 µM/oligo.
      4. If your RNA amount is under NanoDrop detection range, use 0.01 µM/oligo.
    3. Incubate as follows in a PCR machine:
      95 °C for 2 min
      Ramp down to 45 °C at 0.1 °C/sec
      45 °C for 5 min
      Hold at 45 °C
  7. Hybridase® (thermostable RNase H) reaction to digest RNA from RNA:DNA hybrids
    1. Prepare a master mix that you preheat to 45 °C (in a hot block):
      1 μl 10x Hybridase buffer H2 (Recipe 2)
      1 μl Hybridase (5 U/μl)
      2 μl nuclease-free water
    2. Add 4 μl of the MM to hybridization reaction still at 45 °C (keep in the PCR machine).
    3. Incubate at 45 °C for 30 min.
    4. Remove to ice.
  8. DNase I treatment to digest DNA oligos/probes from RNA pool
    1. Use DNase I protocol for Turbo DNase I.
    2. Add to 40 μl of RNA (for four aliquots pooled):
      4 μl of 10x DNase I reaction buffer
      2 μl DNase I
    3. Incubate for 30 min at 37 °C.
    4. Add 2 μl DNase I inactivation reagent (vortex well before adding) and incubate for 5 min at RT (vortex every 1 min). The minimum volume of inactivation reagent to add is 2 μl–so even smaller volumes (e.g., 10 μl), add 2 μl.
    5. Spin down (2,000 x g for 5 min) and recover 43 μl into a new tube.
  9. RNA cleanup using Agencourt RNAClean XP beads
    1. Add 1.8 volume (e.g., 77.4 μl for 43 μl of RNA) of RNAClean XP beads to rxn.
    2. Incubate at RT for 10 min.
    3. Onto a magnet for 5 min.
    4. Remove most of the supernatant (leave 5 μl behind to avoid pulling up beads).
    5. Leave tubes on the magnet, add 200 μl 70% ethanol, let stand for 30 sec.
    6. Remove all the supernatant.
    7. Repeat 70% wash as above once more (two washes total).
    8. Remove as much of the EtOH as possible.
    9. Air dry beads on the magnet for 10 min (until appear dry, see Figure 5).
    10. Add 10 μl RNase-free water to the first aliquot dried bead and mix.
    11. If you have aliquoted samples, pool back together.
    12. Incubate at RT for 5 min.
    13. Onto the magnet for 5 min.
    14. Recover 10 μl eluate. Store RNA at -80 °C.
    Note: The eluate contains your rRNA-free RNA. This is suited for RNA-Seq library construction but remember to skip any polyA-enrichment steps. The RNA-Seq library protocol we used (Reynoso et al., 2018) is the non-strand specific random primed library described by Townsley et al. (2015).

Data analysis

The protocol described above needs to be combined with other downstream protocols (RNA-Seq) to generate data to be analyzed.

Recipes

  1. 5x Hybridization buffer H1 (RNase-free)
    0.5 M Tris
    1 M NaCl
    Adjust pH to 7.0 with HCl
    Filter sterilize through a 0.22 μm filter, aliquot to 1 ml aliquots, and store at -20 °C
  2. 10x Hybridase buffer H2 (RNase-free)
    500 mM Tris
    1 M NaCl
    200 mM MgCl2
    Adjust pH to 7.4 with HCl
    Filter sterilize through a 0.22 μm filter, aliquot to 1 ml aliquots, and store at -20 °C

Acknowledgments

This work was supported by the United States National Science Foundation (NSF) Plant Genome grant No. IOS-1238243 to R.B.D., S.M.B., N.S. and J.B.-S. and by a Finnish Cultural Foundation postdoctoral fellowship (to K.K.). An NSF Research Experiences for Undergraduates supplement (IOS-1238243) and site program (DBI-1461297) supported S.C and J.V., respectively.
Authors declare no conflicts of interest or competing interests.

References

  1. Bajic, M., Maher, K. A. and Deal, R. B. (2018). Identification of open chromatin regions in plant genomes using ATAC-Seq. Methods Mol Biol 1675: 183-201.
  2. Bailey-Serres, J. (2013). Microgenomics: Genome-scale, cell-specific monitoring of multiple gene regulation tiers. Annu Rev Plant Bio 64: 293-325.
  3. Deal, R. B. and Henikoff, S. (2010). A simple method for gene expression and chromatin profiling of individual cell types within a tissue. Dev Cell 18(6): 1030-1040.
  4. Deal, R. B. and Henikoff, S. (2011). The INTACT method for cell type-specific gene expression and chromatin profiling in Arabidopsis thaliana. Nat Protoc 6(1): 56-68.
  5. Morlan, J. D., Qu, K. and Sinicropi, D. V. (2012). Selective depletion of rRNA enables whole transcriptome profiling of archival fixed tissue. PLoS One 7(8): e42882.
  6. Reynoso, M.A, Pauluzzi, G., Kajala, K., Cabanlit, S., Velasco, J., Bazin, J., Deal, R.B., Sinha, N.R., Brady, S.M., Bailey-Serres J. (2018). Nuclear transcriptomes at high resolution using retooled INTACT. Plant Physiol 176(1): 270-281.
  7. Ron, M., Kajala, K., Pauluzzi, G., Wang, D., Reynoso, M. A., Zumstein, K., Garcha, J., Winte, S., Masson, H., Inagaki, S., Federici, F., Sinha, N., Deal, R. B., Bailey-Serres, J. and Brady, S. M. (2014). Hairy root transformation using Agrobacterium rhizogenes as a tool for exploring cell type-specific gene expression and function using tomato as a model. Plant Physiol 166(2): 455-469.
  8. Townsley, B.T., Covington, M.F., Ichihashi, Y., Zumstein, K., Sinha, N.R. (2015). BrAD-seq: Breath Adapter Directional sequencing: a streamlined, ultra-simple and fast library preparation protocol for strand specific mRNA library construction. Front Plant Sci 6: 366.

简介

基因表达在多个水平上动态调节,包括染色质可及性和转录。 为了研究这些核调节事件,我们描述了我们用净化细胞核(INTACT)纯化细胞核的方法。 由于核RNA在聚腺苷酸化转录物中低并且常规下拉方法不会捕获非聚腺苷酸化前mRNA,所以我们还提出了我们的方法以从核RNA总RNA中去除核糖体RNA以准备核RNA-Seq。

【背景】分离用于基因表达实验的特定细胞类型降低了噪音并提高了实验的精确度和发现的不同表达基因的数量。用于细胞类型特异性研究的各种方法被广泛使用,每种方法都有其优点和缺点(Bailey-Serres,2013年综述)。分离特定的调控区室,如细胞核(来自细胞器,核糖体,细胞质,等等)可以进一步精确解析调控基因表达的分子事件。在这里,我们描述了一种方法,可以从冷冻组织中分离特定细胞类型的细胞核,适用于研究核基因表达的实验(例如,核RNA的RNA-Seq,ATAC-Seq,ChIP -Seq,等。)。此外,我们描述了RNA的处理,导致材料适合作为RNA-Seq实验的输入。这里描述的方案与水稻根组织(日本野生稻栽培品种日本晴)(Reynoso等人,2018年)一起使用,但是它们基于以前开发的方案, (拟订和Henikoff,2010年和2011年)和番茄(罗恩等人,2014年)。

该协议的第一部分,INTACT方法(用于标记特定细胞类型的核的分离)允许体内亲和标记和随后从感兴趣的细胞类型中纯化细胞核。这是通过由包膜靶向结构域,GFP和生物素连接酶识别肽(BLRP)组成的三联核标签融合蛋白(NTF)的细胞类型特异性表达实现的。 NTF与E的共同表达。在所关注的细胞类型中密码子优化的大肠杆菌生物素连接酶基因BirA导致在该细胞类型中特异性产生荧光标记的生物素化核。然后可以使用链霉亲和素包被的磁珠从粗制组织匀浆中亲和纯化这些标记的核,从而允许从感兴趣的细胞类型获得RNA和染色质。

该方法的第二部分描述了处理核RNA以产生适合作为RNA-Seq文库制备的输入的样品。通常对于真核样品,通过polyA-下拉方法从RNA样品中去除核糖体RNA(rRNA)和细胞器RNA。然而,核mRNA在许多加工阶段含有前体mRNA,大部分不是聚腺苷酸化的,而许多聚腺苷酸化mRNA迅速输出。因此,为了在处理的不同阶段分析转录本,polyA分离方法不适合。受以前工作的启发(Morlan等人,2012; Gregory Smaldone, personal communication ),我们开发了一种从大米样品中去除核糖体RNA(rRNA)的方法, INTACT纯化的细胞核作为我们的输入。该方法的步骤包括从INTACT纯化的核中分离总RNA,去除残余的基因组DNA,将DNA探针退火至rRNA,用特异于RNA:DNA杂交体的RNA酶降解rRNA,并降解DNA探针。在方案结束时,RNA样品准备好用于RNA-Seq文库构建。

关键字:基因表达, 核纯化, INTACT, rRNA降解, RNA提取, RNA-Seq


第一部分:INTACT纯化核

材料和试剂

  1. 移液器吸头(P20,P200和P1000,无核酸酶,例如,美国科学公司,产品目录号:1123-1710,1120-8780,1126-7510)
  2. (可选)13毫升Falcon管
  3. 30微米细胞过滤器(Sysmex,CellTrics,目录号:04-004-2326)
  4. 15ml Falcon管(无核酸酶,例如VWR,目录号:89039-666)
  5. 1.5ml Eppendorf管(无核酸酶,例如Denville Scientific,目录号:C2170)
  6. 巴斯德移液管(无核酸酶,例如,Phenix Research Products,目录号:PP-137038C)
  7. 幻灯片 
  8. 盖帽 
  9. PCR管带(无核酸酶,例如,美国科学公司,目录号:1402-2708)
  10. 0.22μm注射器过滤器(如,Merck,产品目录号:SLGP033RB)
  11. 50ml Falcon管(无核酸酶,例如VWR,目录号:89039-658)
  12. 铝箔
  13. 具有生物素标记核的转基因植物组织
  14. INTACT二元载体(Ron等人,2014; Reynoso等人,2018)
    注意:它们将可在 https://gateway.psb。 ugent.be/search 。这些质粒允许将您选择的启动子的T-DNA插入您选择的植物物种中。
  15. 液氮
  16. M-280抗生蛋白链菌素Dynabeads(Thermo Fisher Scientific,Invitrogen TM,目录号:11205D)
  17. NaOH
  18. MOPS(Sigma-Aldrich,目录号:M1254-25G)
  19. 氯化钠(NaCl)(例如,Fisher Scientific,目录号:S271)
  20. 氯化钾(KCl)(例如,Fisher Scientific,目录号:BP366-1)
  21. 乙二胺四乙酸(EDTA)(例如,Fisher Scientific,目录号:S311)
  22. EGTA(Sigma-Aldrich,目录号:E3889-25G)
  23. 精胺(Sigma-Aldrich,目录号:S1141)
  24. 亚精胺(Sigma-Aldrich,目录号:S2626)
  25. 蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:P9599)或完全微型蛋白酶抑制剂片剂不含EDTA(Roche Diagnostics,目录号:11836170001)
  26. Triton X-100(Sigma-Aldrich,目录号:648466)
  27. 碘化丙啶(PI)染色(Sigma-Aldrich,目录号:P4170)
  28. 10 N NaOH(见食谱)
  29. 0.5 M MOPS,pH 7(见食谱)
  30. 1 M NaCl(见食谱)
  31. 2 M KCl(见食谱)
  32. 0.5 M EDTA(见食谱)
  33. 0.5米EGTA(见食谱)
  34. 0.2 M精胺(见食谱)
  35. 0.2 M亚精胺(见食谱)
  36. 10%Triton X-100(见食谱)
  37. Nuclei纯化缓冲液碱(见食谱)
  38. NPB和NPBt缓冲液(见食谱)
  39. 碘化丙啶(PI)污渍(见食谱)

设备

  1. 移液器(P20,P200,P1000;例如Eppendorf,产品目录号:3123000039,312000055,3123000063)
  2. 陶瓷研钵和研杵(用RNase Zap [Thermo Fisher Scientific,Invitrogen TM,产品目录号:AM9780]或其他RNase去除产品擦拭干净)
  3. 对15ml Falcon管进行离心(例如,Eppendorf,型号:5810R)。
  4. Dynamag TM -15 Magnet(Thermo Fisher Scientific,型号:Dynamag TM -15,目录号:12301D)
    注:可以用自制版本替换。例如,一个强大的稀土(钕)磁铁(例如,1 x 10厘米长的条形码)可以在50毫升猎鹰管内粘贴,并且可以添加衬垫以保持15毫升Falcon管在磁铁旁边(参见图1 )。


    图1.两支15毫升猎鹰管的自制磁铁两个钕磁铁放置在装在管架上的50毫升Falcon管内。两个磁铁之间的吸引力将它们固定在位。为了支撑15毫升管子抵抗磁铁的位置,纸巾被粘在50毫升管子内。

  5. Dynamag TM-2 Magnet(Thermo Fisher Scientific,型号:Dynamag TM -2,目录号:12321D),或者这个的自制版本。
  6. 血细胞计数器(SCK Films,In-Cyto,产品目录号:DHC-N01-2)
  7. BD Adams TM Nutator TM Single-Speed Orbital Mixer( ,Fisher Scientific,目录号:14-062)
    制造商:BD,目录号:421105 / DEL。
  8. 4°C的冰箱和/或冷藏室
  9. 带滤光片的荧光显微镜可以观察DAPI或PI染色(Edmund Optics,目录号:86-371,67-008)

程序

图2提供了过程的概述,包括可选的检查点。


图2.概述第I部分中的步骤的流程图:INTACT纯化核

注:对于计划使用INTACT纯化细胞核进行染色质免疫沉淀(ChIP)实验(本协议未描述)的研究人员来说:为进行ChIP实验,新鲜组织需要交联。

  1. 通过将所有起始组织放入具有1%甲醛的30ml NPB(Sigma-Aldrich,F8775)在50ml Falcon管中,并将组织在真空下孵育10分钟,将蛋白质交联至DNA。
  2. 将甘氨酸(Fisher Scientific,BP381)加至终浓度为0.125M,置于真空下5分钟以上。
  3. 用水清洗组织3次,并擦干。  

  1. 从冷冻物质中分离核
    1. 根据需要准备新鲜的NPB和NPBt缓冲液(每个样品约15 ml NPB和45 ml NBPt)并在冰上冷却缓冲液。我们建议一次处理最多八个样本。
    2. 在液氮中用陶瓷研钵和杵研磨冷冻组织(50-200厘米1厘米根尖,200根根尖180毫克),直到非常细。
      可选:将粉末倒入13毫升Falcon管中并称重样品。用液氮冷却一切,不要让样品解冻。
    3. 用移液管将约8 ml冰冷的NPB加入研钵中,轻轻研磨以重新悬浮粉状组织(对较大样品按比例放大)。 NPB不会冻结是非常重要的,所以要么使用最少量的液氮进行研磨,要么移动到新的砂浆中进行这一步骤。
    4. 通过30μm细胞过滤器将悬浮液过滤到15 ml Falcon管中,并在处理其余样品时将Falcon管置于冰上。

    5. 在4°C(在Eppendorf 5810 R离心机上以2,229 rpm)离心过滤的细胞核1000 x g,15分钟。
    6. 准备在1.5毫升管(每个样品一个)的链霉亲和素珠。为每个样品清洗25μlInvitrogen M-280 Streptavidin Dynabeads。为了清洗珠子,将充分混合的珠子移入1.5ml管中,放置在磁体上,除去上清液,加入并与1ml NPB混合,放置在磁体上,除去上清液,重新悬浮在原始体积的NPB中(50μl )。
    7. 将Falcon管从离心机移至冰上。
      可选:标记上清液(SN)的体积并将10%的上清液用于蛋白质印迹('SN1000' - 如果您想确认您的上清液中没有丢失任何细胞核/生物素信号)。
      1. 弃去上清液(小心倒入)。
      2. 通过上下吸取将1ml冷NPB中的细胞核重新悬浮。换句话说,在核颗粒上从移液管释放缓冲液直到颗粒完全重新悬浮。
      3. 留出11μl重悬的核用于定量和产量计算(PCR条效果良好)。见程序B.
        可选:为Western印迹(样品中生物素信号的总量)预留10%重悬'P1000'(Pellet)。
    8. 将重新悬浮的细胞核(〜1ml)转移到洗涤过的珠上(在1.5ml管中)。在4°C的条件下,在nutator上旋转管(约20 rpm)30分钟,将核膜包被的生物素结合到珠子上的链霉亲和素上。
    9. 在15ml Falcon管中用14ml NPBt稀释1ml珠 - 核混合物。轻轻混合并置于4°C的章动器(约20 rpm)30秒。
      在4°C放置磁铁15-20分钟。
      可选:预留10%的上清液进行Western印迹('Unbound')。
    10. 用巴氏吸管小心地取出上清液,轻轻地将悬浮液重悬于14ml NPBt中。轻轻混合并置于4°C的章动器(约20 rpm)30秒。
      将管子放在磁铁上10分钟(4°C)。
    11. 重复步骤A10。
    12. 轻轻去除上清液。在1毫升NPBt中重悬珠。
      预留11微升核用于定量和产量计算(PCR条效果良好)。见程序B.
      可选:将10%的重悬微珠用于蛋白质印迹(“结合”以确保大部分生物素化信号在此处)。
    13. 将1ml重悬液转移到1.5ml管中并捕获在磁体上。
    14. 取出上清液,用20μlNPB重悬珠。
      注意:只有在计数和等分后,才能冻结核。

  2. 用血细胞计数器计数核以量化产量
    1. 用0.5μl10μg/ ml碘化丙啶(PI)染色混合11μl细胞核(来自步骤A14的珠粒或来自步骤A7c的重悬浮丸粒)。成像前,让细胞核染色至少5分钟。
    2. 吸取10微升样品到血球计上。一个血细胞计数器有两个位置,A和B,用于计数原子核,因此您需要一个血球计数器来计数两个样品。将样品移液到半月形区域,推动载玻片和盖玻片之间的细胞核再悬浮(图3)。
    3. 在荧光灯下观察样品并使用适合PI的过滤器,使用计数网格对核进行计数(图3)。用它来计算总样品中的核数。 


      图3.血细胞计数器上计数网格的外观,以及用共焦激光扫描显微镜观察PI染色的细胞核

      1. 例如,您可以在网格的角落计算16个方格,包括整个网格的1/9(该面积为1 x 1 x 0.1 mm或0.1μl,并且代表1 ml的1/100)。
      2. 将该区域内发现的细胞核数量乘以10,000,找出总产量(您分离出多少个细胞核)。
        建议:
        1. 来自200个根尖的强的近组成型启动子系(例如35Spro:NTF)的典型产量为150,000-230,000个核。
        2. 检查/计数总核数的起始数(使用来自步骤A7c的细胞核),如果您对显微镜下原始细胞核的分离和细胞核的外观有疑虑的话。
      3. 可选计算:通过使用非转基因组织('阴性对照')和与组成型启动子('35S-IN')相比较,使用原子核中拉出的细胞核和总原子核数目(P1000 ):
        效率:( 35S-IN珠 - 背景)/(35S-IN颗粒)
        背景:(35S-IN珠)×(阴性对照珠)/(阴性对照颗粒) 

  3. 核糖核苷酸和ATAC的等分。
    注意:除了RNA-Seq之外,只有在使用分离的细胞核进行ATAC-seq实验(由Bajic等人,2018详细描述)时,样品的分裂才是相关的。
    1. 计算您需要从最终的20μl中获得多少μl的核,以获得50,000个ATAC核。将它们吸到冰上的PCR条上并开始ATAC方案而不冻结(Bajic et al。 ,2018)。
    2. 立即使用RNA的其余部分进行RNA,或在-80°C下冷冻以备后续RNA工作。

数据分析

上述方案需要与其他下游方案(RNA-Seq,ATAC-Seq,ChIP-Seq,qRT-PCT)结合以产生待分析的数据。

食谱

  1. 10 N NaOH
    向80ml dH 2 O中缓慢加入40g氢氧化钠颗粒,同时不断搅拌。
  2. 0.5 M MOPS pH 7

    20.93克MOPS在200毫升去离子水中
    用10N NaOH(或NaOH颗粒)调节pH至7.0 过滤器通过0.22微米过滤器消毒,等分40毫升等分50毫升猎鹰管,并存储在-20°C
  3. 1 M NaCl

    在200毫升去离子水中含11.688克NaCl 高压灭菌器,在室温下储存
  4. 2 M KCl

    29.8205克氯化钾溶于200毫升去离子水中 高压灭菌器,在室温下储存
  5. 0.5 M EDTA

    在200毫升去离子水中的29.224克EDTA 用固体NaOH(> 8g)调节pH至8.0 注意:在pH值调整之前,EDTA不溶解。
    高压灭菌器,在室温下储存
  6. 0.5米EGTA

    19.0175克EGTA溶于100毫升去离子水中 用固体NaOH(约4克)调整pH8.0。
    注意:EGTA不会溶解,直到pH值调整完毕。
    高压灭菌器,在室温下储存
  7. 0.2 M精胺
    1克在24.7毫升去离子水中
    过滤器通过0.22μm过滤器消毒,分装,并在-20°C冷冻。
  8. 0.2 M亚精胺
    1克在34.4毫升去离子水中。
    过滤器通过0.22μm过滤器消毒,分装,并在-20°C冷冻。
  9. 10%Triton X-100
    5毫升Triton X-100

    45毫升无核酸酶水 保持室温
  10. 核纯化缓冲液(NPB)基地
    NPB基质可以通过0.22μm的过滤器过滤灭菌,并保持在4℃达3个月。每个样品通常需要60ml的NPB碱基。
    20 mM MOPS(pH 7)
    40 mM NaCl
    90 mM KCl
    2 mM EDTA
    0.5 mM EGTA
    对于60毫升(一个样品):
    2.4毫升0.5 M MOPS
    2.4毫升1 M NaCl
    2.7毫升2 M KCl
    240μl0.5M EDTA pH 8
    60μl0.5M EGTA pH 8
    1 L(〜16个样本):
    40毫升0.5 M MOPS
    8毫升5 M NaCl
    45毫升2 M KCl
    4毫升0.5M EDTA pH 8
    1ml 0.5M EGTA pH 8
  11. NPB和NPBt缓冲液

    在使用前加入Triton X-100,亚精胺,精胺和完全蛋白酶抑制剂,并保存在冰上或4°C。
  12. 碘化丙啶(PI)染色

    通过将10毫克PI溶解在1毫升蒸馏水中制备1,000倍浓缩储备溶液
    使用蒸馏水1:1,000稀释10μg/ ml PI的1x工作溶液 将溶液保存在4°C,用铝箔包裹以防止降解

第二部分:总RNA分离和rRNA降解

材料和试剂

  1. 移液器吸头(用于P10,P20,P200,P1000,无核酸酶例如,USA Scientific,目录号:1123-1710,1120-8780,1126-7510)
  2. 1.5ml Eppendorf管(无核酸酶,例如Denville Scientific,目录号:C2170)
  3. 无RNase的PCR条(例如,美国科学公司,产品目录号:1402-2708)
  4. 0.22μm注射器过滤器(如,Merck,产品目录号:SLGP033RB)
  5. 在第一部分用INTACT分离的核,或任何其他合适的起始组织。
  6. 针对rRNA序列设计的1μMDNA探针库存
    注意:请参阅有关如何在过程的第1步中设计这些内容的详细说明。
  7. RNeasy Micro Kit(QIAGEN,目录号:74004)或RNeasy Micro Plus Kit(QIAGEN,目录号:74034)
    注意:这两个套件都包含所需的组件,但通常一个套件比另一套更便宜。
  8. 70%乙醇
  9. 无RNase水
  10. Turbo DNA酶试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:AM2238)
  11. Agencourt RNAClean XP珠(Beckman Coulter,目录号:A66514)
    或者:使用AMPureXP珠(Beckman Coulter,目录号:A63881)。
  12. 杂交酶耐热RNA酶H(Epicentre,目录号:H39500)
  13. Tris碱(例如,Fisher Scientific,目录号:BP154-1)
  14. 盐酸(HCl,例如,Sigma-Aldrich,目录号:435570)
  15. 氯化钠(NaCl)(例如,Fisher Scientific,目录号:S271)
  16. 氯化镁(MgCl 2)(例如,Sigma-Aldrich,目录号:M8266)
  17. 5倍杂交缓冲液H1(见食谱)
  18. 10x Hybridase缓冲液H2(见食谱)

设备

  1. 移液器(P20,P200,P1000;例如Eppendorf,产品目录号:3123000039,312000055,3123000063)
  2. Dynamag TM-2 Magnet(Thermo Fisher Scientific,型号:Dynamag TM -2,目录号:12321D),或者这个的自制版本。
  3. 用于PCR条带的磁性架( ,Edge BioSystems,产品目录号:57624)
  4. 微量离心机(例如,Eppendorf,目录号:5424系列)
  5. PCR仪(例如,Thermo Fisher Scientific,Applied Biosystems TM,型号:2720)。
  6. NanoDrop分光光度计(例如,Thermo Fisher Scientific,Thermo Scientific TM,型号:NanoDrop TM 2000,目录号:ND-2000) >

软件

  1. 序列分析软件:SnapGene,Geneious,等

程序

图4提供了该协议的概述,包括所有可能的停止点和温度(理论上,您可以在这些点停留多达几周)。


图4.总结第二部分中的步骤的流程图:总RNA分离和rRNA降解

  1. 设计针对rRNA序列的DNA探针
    1. 设计针对rRNA序列的DNA探针是此过程的关键部分,因为这是您针对RNA降解的方法。为了靶向核rRNA,您需要获得5S,5.8S,18S和25S rRNA及其内部和外部转录间隔子的序列。如果您打算对总细胞RNA进行rRNA降解,请记住也针对线粒体和质体rRNA。对于许多物种,这些序列可以从 https://www.ncbi.nlm.nih。 gov / nucleotide /
    2. 设计您的探针长度为60 bp,覆盖整个rRNA的长度(包括间隔区,因为它们将存在于核rRNA中),并反向补充rRNA序列。任何能够将序列转换成反向互补的序列分析软件(SnapGene,Geneious,等等)都可以使用。
    3. 订购你的探针(DNA寡核苷酸)作为预先制成的混合物在1000纳升水10 nmol。这是您的10μM库存。
    4. 将您的股票10x稀释成工作混合物,其中每个探针的浓度均为1μM。
  2. QIAGEN RNeasy Micro Kit通过使用INTACT分离的细胞核进行总RNA分离
    如下使用QIAGEN RNeasy Micro Kit纯化核RNA:
    1. 首先向20μl纯化的核中加入350μl裂解缓冲液RLT(加入10μlβ-巯基乙醇/ 1ml)。剧烈漩涡2分钟。
    2. 在室温下将溶解产物在1,000xg(3,100rpm)离心2分钟以沉淀珠子。将管置于Dynamag TM TM-2磁体上,并将上清液转移到新的1.5ml管中。加入350μl70%乙醇并涡旋数次混匀。
    3. 将溶胞产物/乙醇混合物移入放置在2ml收集管中的RNeasy MinElute离心柱中,并在室温下以10,000xg(9,700rpm)离心1分钟。丢弃流量。
    4. 将350μl缓冲液RW1加入色谱柱。在室温以10,000 em x g(9,700 rpm)离心1分钟。丢弃流通并将色谱柱移至新的2 ml收集管。
    5. 添加500μL缓冲RPE的列。在室温以10,000 em x g(9,700 rpm)离心1分钟。丢弃流量。
    6. 向柱中加入500μl80%乙醇。在室温以10,000 em x g(9,700 rpm)离心1分钟。丢弃流通并将色谱柱移至新的2 ml收集管。
    7. 打开柱盖并在室温下以最高速度离心(16,000×g克)5分钟。丢弃流通液并将色谱柱放入新的1.5 ml管中。
    8. 向柱膜上加20μl无RNase的水,静置1分钟。
      在16,000×g g离心1分钟
    9. 将RNA保存在-80°C。
  3. DNA酶I治疗以消除基因组DNA污染
    使用DNase I协议用于Turbo DNase I,如下所示:
    1. 加入20μl的RNA:
      2μl10x DNA酶I反应缓冲液
      1μlDNase I

    2. 在37°C孵育30分钟
    3. 加入2μlDNase I灭活试剂(加入前充分涡旋)并在室温孵育5分钟(每1分钟涡旋一次)。
    4. 旋转(2000×g <5分钟)并将20μl回收到新管中。
  4. 使用Agencourt RNAClean XP珠去除DNase缓冲液进行RNA清除
    1. 将RNAClean XP珠粒加入1.8倍体积(例如,36μl用于20μlRNA)至反应中。
    2. 在室温孵育10分钟。
    3. 在磁铁上5分钟。
    4. 去除大部分上清液(留下5μl以避免吸取珠子)。
    5. 将管置于磁铁上,加入200μl70%乙醇,静置30秒。
    6. 去除所有上清液。

    7. 重复70%以上的洗涤次数(总共洗两次)。

    8. 尽可能多地去除EtOH。
    9. 空气干燥磁珠10分钟(直到干燥,见图5)。

    10. 加入15μl无RNase的水至第一份干燥珠粒并混匀。
    11. 如果你有等分样品,请一起回来。
    12. 在室温孵育5分钟。
    13. 在磁铁上5分钟。
    14. 回收15μl洗脱液。


      图5. Agencourt XP珠子的外观。 :一种。除去乙醇后,珠粒会出现闪亮和潮湿。 B.准备洗脱的珠子看起来干燥,颗粒较细,颜色通常较浅。

  5. NanoDrop for [RNA]和260/280,以便您知道用于调整探针浓度的RNA浓度。
  6. rRNA探针杂交使DNA探针与rRNA结合
    1. 步骤6和步骤7的反应大小需要为6μl+4μl(总共10μl),以使缓冲液对于每一步都是最佳的。在DNase I处理前(步骤8),您可以制备多个反应等分试样并回收。
      成功的RNA-Seq文库已由单一0.1μg反应制成
    2. 将以下内容加在一起:
      3.8微升RNA(如果这将超过1微克RNA,弥补无RNase水)
      1.2μl5x杂交缓冲液H1(配方1)
      1.0μl探针混合物 - 选择您的浓度如下:
      1. 如果您的RNA量为1μg,则使用1μM/寡核苷酸探针混合物。
      2. 如果您的RNA量为0.1μg,则将探针稀释至0.1μM/ oligo。
        注意:这是我们实验中最常见的探针浓度。
      3. 如果您的RNA量为0.01μg,则将探针稀释至0.01μM/ oligo。
      4. 如果您的RNA量在NanoDrop检测范围内,请使用0.01μM/ oligo。
    3. 在PCR仪器中孵育如下:
      95°C 2分钟

      以0.1°C /秒降至45°C 45°C 5分钟

      保持在45°C
  7. 杂交酶(热稳定RNA酶H)反应以消化来自RNA:DNA杂合体的RNA
    1. 准备一个预混到45°C(在热块中)的主混合物:
      1μl10x Hybridase缓冲液H2(方案2)
      1μl杂交酶(5 U /μl)
      2μl无核酸酶水

    2. 在45°C条件下,将4μl的MM加入到杂交反应中(保留在PCR仪中)。

    3. 在45°C孵育30分钟
    4. 去冰。
  8. DNase I处理从RNA池中消化DNA寡核苷酸/探针
    1. 为DNase I使用DNase I协议。
    2. 加入40μlRNA(四份合并):
      4μl的10x DNase I反应缓冲液
      2μlDNase I

    3. 在37°C孵育30分钟
    4. 加入2μlDNase I灭活试剂(加入前充分涡旋)并在室温孵育5分钟(每1分钟涡旋)。添加的灭活试剂的最小体积是2μl,因此甚至更小的体积(例如10μl)加入2μl。
    5. 旋转(2,000 em x g <5分钟)并将43μl回收到新的试管中。
  9. 使用Agencourt RNAClean XP珠子进行RNA清除
    1. 将RNAClean XP珠的1.8倍体积(例如,77.4μl用于43μlRNA)添加到rxn。
    2. 在室温孵育10分钟。
    3. 在磁铁上5分钟。
    4. 去除大部分上清液(留下5μl以避免提起珠子)。
    5. 将管置于磁铁上,加入200μl70%乙醇,静置30秒。
    6. 去除所有上清液。

    7. 重复70%以上的洗涤次数(总共洗两次)。

    8. 尽可能多地去除EtOH。
    9. 空气干磁珠10分钟(直到干燥,见图5)。
    10. 向第一份等分试样干珠中加入10μl不含RNase的水并混合。
    11. 如果你有等分样品,请一起回来。
    12. 在室温孵育5分钟。
    13. 在磁铁上5分钟。
    14. 回收10μl洗脱液。将RNA保存在-80°C。
    注意:洗脱液含有无rRNA的RNA。这适用于RNA-Seq文库构建,但请记住跳过任何polyA富集步骤。我们使用的RNA-Seq文库方案(Reynoso等,2018)是Townsley等人描述的非链特异性随机引物文库。 (2015)。

数据分析

上述协议需要与其他下游协议(RNA-Seq)相结合以生成待分析的数据。

食谱

  1. 5x杂交缓冲液H1(无RNase)
    0.5 M Tris
    1 M NaCl
    用HCl调节pH至7.0
    通过0.22μm过滤器过滤消毒,等分至1 ml等分试样,并储存于-20°C。
  2. 10x Hybridase缓冲液H2(无RNase)
    500 mM Tris
    1 M NaCl
    200mM MgCl 2 2/2 用HCl调节pH至7.4
    通过0.22μm过滤器过滤消毒,等分至1 ml等分试样,并储存于-20°C。

致谢

这项工作得到美国国家科学基金会(NSF)植物基因组授权号IOS-1238243对R.B.D.,S.M.B.,N.S.的支持。和J.B.-S.和芬兰文化基金会博士后奖学金(K.K.)。美国国家科学基金会本科生补充研究经历(IOS-1238243)和现场项目(DBI-1461297)分别支持S.C和J.V.。
作者声明不存在利益冲突或利益冲突。

参考

  1. Bajic,M.,Maher,K.A。和Deal,R.B。(2018)。 使用ATAC-Seq识别植物基因组中的开放染色质区域 <方法Mol Biol 1675:183-201。
  2. Bailey-Serres,J.(2013)。 Microgenomics:多基因调控层的基因组规模,细胞特异性监测 Annu Rev Plant Bio 64:293-325。
  3. Deal,R.B.和Henikoff,S.(2010)。 一种用于组织内单个细胞类型的基因表达和染色质分析的简单方法。 Dev Cell 18(6):1030-1040。
  4. Deal,R.B.和Henikoff,S.(2011)。 在拟南芥中用于细胞类型特异性基因表达和染色质分析的INTACT方法 em>。 Nat Protoc 6(1):56-68。
  5. Morlan,J. D.,Qu,K.和Sinicropi,D.V。(2012)。 rRNA的选择性消耗可以实现档案固定组织的全部转录组分析 PLoS一个 7(8):e42882。
  6. Reynoso,M.A,Pauluzzi,G.,Kajala,K.,Cabanlit,S.,Velasco,J.,Bazin,J.,Deal,R.B.,Sinha,N.R.,Brady,S.M.,Bailey-Serres J.(2018)。 使用重组INTACT的高分辨率核转录组 Plant Physiol 176(1):270-281。
  7. Ron,M.,Kajala,K.,Pauluzzi,G.,Wang,D.,Reynoso,MA,Zumstein,K.,Garcha,J.,Winte,S.,Masson,H.,Inagaki,S.,Federici ,F.,Sinha,N.,Deal,RB,Bailey-Serres,J.和Brady,SM(2014)。 使用毛根土壤杆菌作为探索细胞类型的工具的毛状根转化特定的基因表达和功能使用番茄作为模型。植物生理学166(2):455-469。
  8. Townsley,B.T.,Covington,M.F.,Ichihashi,Y.,Zumstein,K.,Sinha,N.R.(2015年)。 BrAD-seq:呼吸适配器定向测序:简化,超简单,快速的文库准备方案 链特异性mRNA文库构建。 前植物科学 6:366.
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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: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Reynoso, M. A., Pauluzzi, G. C., Cabanlit, S., Velasco, J., Bazin, J., Deal, R., Brady, S., Sinha, N., Bailey-Serres, J. and Kajala, K. (2018). Isolation of Nuclei in Tagged Cell Types (INTACT), RNA Extraction and Ribosomal RNA Degradation to Prepare Material for RNA-Seq. Bio-protocol 8(7): e2458. DOI: 10.21769/BioProtoc.2458.
  2. Reynoso, M.A, Pauluzzi, G., Kajala, K., Cabanlit, S., Velasco, J., Bazin, J., Deal, R.B., Sinha, N.R., Brady, S.M., Bailey-Serres J. (2018). Nuclear transcriptomes at high resolution using retooled INTACT. Plant Physiol 176(1): 270-281.
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