Simultaneous Profiling of Chromosome Conformation and Gene Expression in Single Cells
同时分析单细胞染色体构象和基因表达
(*contributed equally to this work) 发布: 2023年11月20日第13卷第22期 DOI: 10.21769/BioProtoc.4886 浏览次数: 1540
评审: Rajesh RanjanVartika SharmaAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jun 2023
Abstract
Rapid development in single-cell chromosome conformation capture technologies has provided valuable insights into the importance of spatial genome architecture for gene regulation. However, a long-standing technical gap remains in the simultaneous characterization of three-dimensional genomes and transcriptomes in the same cell. We have described an assay named Hi-C and RNA-seq employed simultaneously (HiRES), which integrates in situ reverse transcription and chromosome conformation capture (3C) for the parallel analysis of chromatin organization and gene expression. Here, we provide a detailed implementation of the assay, using mouse embryos and cerebral cortices as examples. The versatility of this method extends beyond these two samples, with the potential to be used in various other cell types.
Key features
• A multi-omics sequencing approach to profile 3D genome structure and gene expression simultaneously in single cells.
• Compatible with animal tissues.
• One-tube amplification of both DNA and RNA components.
• Requires three days to complete.
Graphical overview
Schematic illustration for the Hi-C and RNA-seq employed simultaneously (HiRES) workflow
Hi-C (Hi-C)
RNA-seq (RNA-seq)
Multi-omics (多组学)
Three-dimensional genome (三维基因组)
Transcriptome (转录组)
Background
The acquisition of distinct cellular identities in multicellular organisms during development requires precise regulation of gene expression across various levels. The progress of single-cell multi-omics sequencing technologies has provided new ways for the comprehensive profiling of the transcriptome in conjunction with various other features, including DNA methylation, cellular proteins, chromatin accessibility, and histone modifications [1–4]. Emerging evidence has suggested that transcription is also closely related with higher-order spatial organization of chromatin in the nucleus [5–7]. Nevertheless, there is a lack of techniques allowing for simultaneous profiling of chromatin conformation and gene expression in individual cells. We have recently developed a method named Hi-C and RNA-seq employed simultaneously (HiRES) [8]. Here, we provide a step-by-step guide on how to perform the assay on mouse embryos and brains. This assay can also be performed on other cell lines or tissues from which single-cell suspensions can be obtained.
Materials and reagents
Materials
Tools and supplies for animal dissection (e.g., forceps, tweezers, eye scissors)
DNA low-bind disposable tips (Axygen, catalog numbers: TF-1000-L-R-S, TF-200-L-R-S, TF-20-L-R-S, TF-300-L-R-S)
Multi-channel pipette tips (Rainin, catalog numbers: 30374581, 30374584)
DNA LoBind tube 1.5 mL (Eppendorf, catalog number: 022431021)
0.5 mL thin-wall PCR tubes (Axygen, catalog number: PCR-05-C)
Thermo-fast 96-well plates (Thermo Fisher, catalog number: AB-1400-L)
40 μm cell strainer (Falcon, catalog number: 352340)
0.22 μm filter unit, sterile (Millipore, catalog number: SLGP033RB)
Hemacytometer (INCYTO, catalog number: DHC-N015)
Dounce tissue grinder, 1 mL (Wheaton, catalog number: 357538)
DNA Clean & Concentrator-5 (Zymo Research, catalog number: D4014)
AMPure XP beads (Beckman Coulter, catalog number: A63881)
Adhesive PCR plate seal (Bio-Rad, catalog number: MSB1001)
Reagents
RNase ZapTM (Invitrogen, catalog number: AM9780)
0.4% Trypan Blue solution (Sigma, catalog number: T8154-20ML)
Nuclease-free water (not DEPC-treated) (Thermo Fisher, catalog number: AM9937)
Phosphate-buffered saline (PBS) (Thermo Fisher, catalog number: 10010049)
Fetal bovine serum (FBS) (Gibco, catalog number: 10099141C)
TrypLETM Express (Gibco, catalog number: 12604013)
Sucrose, reagent grade, 99% (Sigma, catalog number: V900116-500G)
HEPES, BioUltra (Sigma, catalog number: 54457-250G-F)
1 M potassium chloride (KCl) (Sigma, catalog number: 60142-100ML-F)
1 M magnesium chloride (MgCl2) (Thermo Fisher, catalog number: AM9530G)
0.1 M DL-Dithiothreitol solution (DTT) (Invitrogen, catalog number: 18090050)
RNase inhibitor (TaKaRa, catalog number: 2313B)
SUPERaseInTM RNase inhibitor (Invitrogen, catalog number: AM2694)
10% Igepal CA 630 (Sigma, catalog number: I8896-100ML)
Protease inhibitor cocktail (Sigma, catalog number: P8340-5ML)
16% formaldehyde solution (Thermo Fisher, catalog number: 28906)
Bovine serum albumin, powder (BSA) (Sigma, catalog number: B2064)
Bovine serum albumin (20 mg/mL BSA) (NEB, catalog number: B9000S)
Glycerol (Sigma, catalog number: 49767-250ML)
1 M Tris pH 8.0 (Invitrogen, catalog number: AM9855G)
5 M sodium chloride (NaCl) (Invitrogen, catalog number: AM9760G)
Sodium dodecyl sulfate solution, BioUltra, ~10% in H2O (10% SDS) (Sigma, catalog number: 71736-100ML)
TritonTM X-100 solution, BioUltra, ~10% in H2O (10% Triton X-100) (Sigma, catalog number: 93443)
10 mM deoxynucleotide (dNTP) solution mix (dNTPs) (NEB, catalog number: N0447L)
100 mM magnesium sulfate solution (MgSO4) (NEB, catalog number: M0259L)
Betaine solution, 5 M, PCR reagent (Sigma, catalog number: B0300-5VL)
5× SuperScriptTM IV RT buffer (Invitrogen, catalog number: 18090050)
SuperScriptTM IV reverse transcriptase (Invitrogen, catalog number: 18090050)
10× NEBuffer 2 (NEB, catalog number: B7002S)
25 U/μL MboI (NEB, catalog number: R0147M)
10× T4 DNA ligase reaction buffer (NEB, catalog number: B0202S)
T4 DNA ligase (1 U/μL) (Invitrogen, catalog number: 15224-025)
QIAGEN protease (30 AU) (QIAGEN, catalog number: 19157)
0.5 M Ethylenediaminetetraacetic acid, pH 8.0 (EDTA) (Invitrogen, catalog number: AM9260G)
10× ThermoPol® reaction buffer (NEB, catalog number: M0259L)
Deep Vent® (exo-) DNA polymerase (NEB, catalog number: M0259L)
TruePrep DNA Library Prep kit V2 for Illumina®, 5× TTBL (Vazyme, catalog number: TD501)
TruePrep DNA Library Prep kit V2 for Illumina®, TTE Mix V50 (Vazyme, catalog number: TD501)
KAPA HiFi PCR Kit, 5× KAPA HiFi GC buffer (Roche, catalog number: KK2102)
KAPA HiFi PCR Kit, KAPA HiFi DNA polymerase (1 U/μL) (Roche, catalog number: KK2102)
High Sensitivity NGS Fragment Analysis kit (Agilent, catalog number: DNF-474-0500)
i5 and i7 unique dual (UD) index (Illumina, catalog numbers: 20091654, 20091656, 20091658, 20091660)
Primers (synthesized by Sangon Biotech with PAGE purification)
GAT5-RT: GTAGGTGTGAGTGATGGTTGAGGTAGTATTGCGCAATGNNNNNNNN TTTTTTTTTTTTTTTVN
GAT5-7N: GTAGGTGTGAGTGATGGTTGAGGTAGTNNNNNNN
GAT5: GTAGGTGTGAGTGATGGTTGAGGTAGT
RNA-P7: GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGTTGAGGTAGT ATTGCGCAATG
Solutions
2% BSA (see Recipes)
1.5 M Sucrose (see Recipes)
60 mg/mL Qiagen protease (see Recipes)
100 mM HEPES buffer pH 8.0 (see Recipes)
2× SC lysis buffer (see Recipes)
Recipes
2% BSA (5 mL)
Reagent Final concentration Quantity 1 M Tris-HCl pH 8.0
1 M KCl
500 mM EDTA
H2O
BSA (powder)
40 mM
200 mM
0.2 mM
n/a
2% (w/v)
200 μL
1 mL
2 μL
3,798 μL
0.2 g
Total n/a 5 mL * *Note: Filter through a 0.22 μm filter. Add an equal volume of glycerol and store at -20 °C (for long-term storage, keep at -80 °C).
1.5 M sucrose (10 mL)
Reagent Final concentration Quantity Sucrose
H2O
1.5 M
n/a
5.13 g
10 mL
Total n/a 10 mL * *Note: Filter through a 0.22 μm filter. Aliquot and store at -20 °C. H2O should be added in increments of 10 mL.
60 mg/mL Qiagen protease
Reagent Final concentration Quantity 7.5 AU Qiagen protease
H2O
60 mg/mL
n/a
One vial
2.78 mL
Total n/a 2.78 mL * *Note: The minimum package volume to be purchased was 7.5 AU/tube. Add water to dissolve directly. Mix well and filter through a 0.2 μm filter. Aliquot and store at 4 °C (maximum storage time is two months).
100 mM HEPES buffer pH 8.0 (10 mL)
Reagent Final concentration Quantity HEPES
H2O
100 mM
n/a
0.24 g
see Note*
Total n/a 10 mL * *Note: First, add 9 mL of H2O. Allow the solution to mix completely before adding more. At this time, the liquid is acidic; adjust the pH of the solution to 8.0 with 1 M NaOH and then bring the volume up to 10 mL with H2O. Filter through a 0.22 μm filter. Aliquot and store at -20 °C.
2× SC lysis buffer (5 mL)
Reagent Final concentration Quantity 1 M Tris-HCl pH8.0
5 M NaCl
10% Triton X-100
500 mM EDTA
1 M DTT
H2O
40 mM
100 mM
0.3% (v/v)
2 mM
50 mM
n/a
200 μL
100 μL
150 μL
20 μL
250 μL
4,280 μL
Total n/a 5 mL * *Note: Aliquot and store at -20 °C.
Equipment
Eppendorf ThermoMixer® C (Eppendorf, model: ThermoMixer C)
Vortex mixer (Scientific Industries, model: Vortex-Genie 2)
Centrifuge (Eppendorf, model: 5424)
Tube revolver rotator (Thermo, model: 88881002)
Thermocycler (Bio-Rad, model: C1000)
Magnetic separator (Invitrogen, model: DynaMagTM -2)
Fluorometer (Invitrogen, model: Qubit 4)
Flow cytometer (BD FACSAriaTM Cell Sorter, model: 65011040)
Bioanalyzer (Agilent Fragment Analyzer, model: 5200)
Software and datasets
Snakemake (v5.20.1, access date: Oct 19, 2023) [9] (Conda: https://anaconda.org/bioconda/snakemake)
hickit (v0.1.1, access date: Oct 19, 2023) (GitHub: https://github.com/lh3/hickit/)
CHARMtools (v0.1, access date: Oct 19, 2023) (GitHub: https://github.com/skelviper/CHARMtools)
Other software or packages listed in https://github.com/skelviper/HiRES/blob/main/HiRES_preprocess_pipeline/envs/main_env.yaml
Procedure
文章信息
版权信息
© 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Chen, Y., Xu, H., Liu, Z. and Xing, D. (2023). Simultaneous Profiling of Chromosome Conformation and Gene Expression in Single Cells. Bio-protocol 13(22): e4886. DOI: 10.21769/BioProtoc.4886.
- Liu, Z., Chen, Y., Xia, Q., Liu, M., Xu, H., Chi, Y., Deng, Y. and Xing, D. (2023). Linking genome structures to functions by simultaneous single-cell Hi-C and RNA-seq. Science 380(6649): 1070–1076. doi: 10.1126/science.adg3797
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分类
系统生物学 > 基因组学 > 测序
分子生物学 > DNA > DNA 测序
细胞生物学 > 单细胞分析
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