Oct 18, 2024

Public workspaceDroplet Hi-C for Fast and Scalable Profiling of Chromatin Architecture in Single Cells

This protocol is a draft, published without a DOI.
  • Lei Chang1,
  • Yang Xie1,2,
  • Brett Taylor1,2,3,
  • Zhaoning Wang1,
  • Jiachen Sun4,5,
  • Ethan J. Armand1,6,
  • Shreya Mishra4,
  • Jie Xu1,
  • Melodi Tastemel1,
  • Audrey Lie1,
  • Zane A. Gibbs1,
  • Hannah S. Indralingam1,
  • Tuyet M. Tan7,
  • Rafael Bejar7,
  • Clark C. Chen8,
  • Frank B. Furnari9,
  • Ming Hu4,
  • Bing Ren1,7,10
  • 1Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA, USA;
  • 2Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, CA, USA;
  • 3Medical Scientist Training Program, University of California, San Diego, La Jolla, CA, USA;
  • 4Department of Quantitative Health Sciences, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, USA;
  • 5Systems Biology and Bioinformatics PhD Program, Case Western Reserve University School of Medicine, Cleveland, OH, USA;
  • 6Bioinformatics and Systems Biology Program, University of California, San Diego, La Jolla, CA, USA;
  • 7Moores Cancer Center, UC San Diego, La Jolla, CA, USA;
  • 8Department of Neurosurgery, University of Minnesota, Minneapolis, MN, USA;
  • 9Department of Medicine, University of California, San Diego School of Medicine, La Jolla, CA, USA;
  • 10Center for Epigenomics, Institute for Genomic Medicine, School of Medicine, University of California, San Diego, La Jolla, CA, USA
Lei Chang
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External link: https://doi.org/10.1038/s41587-024-02447-1
Protocol Citation: Lei Chang, Yang Xie, Brett Taylor, Zhaoning Wang, Jiachen Sun, Ethan J. Armand, Shreya Mishra, Jie Xu, Melodi Tastemel, Audrey Lie, Zane A. Gibbs, Hannah S. Indralingam, Tuyet M. Tan, Rafael Bejar, Clark C. Chen, Frank B. Furnari, Ming Hu, Bing Ren 2024. Droplet Hi-C for Fast and Scalable Profiling of Chromatin Architecture in Single Cells. protocols.io https://protocols.io/view/droplet-hi-c-for-fast-and-scalable-profiling-of-ch-dpxe5pje
Manuscript citation:
Chang, L., Xie, Y., Taylor, B. et al. Droplet Hi-C enables scalable, single-cell profiling of chromatin architecture in heterogeneous tissues. Nat Biotechnol (2024). https://doi.org/10.1038/s41587-024-02447-1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 18, 2024
Last Modified: October 18, 2024
Protocol Integer ID: 110278
Keywords: Single-cell Hi-C, high throughput scHi-C, single-cell sequencing
Funders Acknowledgement:
Bing Ren
Grant ID: 1UM1HG011585
Ming Hu
Grant ID: 1UM1HG011585
Ming Hu
Grant ID: R35HG011922
Frank B. Furnar
Grant ID: R56 NS080939
Frank B. Furnar
Grant ID: R01 CA258248
Brett Taylor
Grant ID: 5T32CA067754-27
Zane A. Gibbs
Grant ID: 5T32CA067754-27
Abstract
Comprehensive analysis of chromatin architecture is crucial for understanding the gene regulatory programs during development and in disease pathogenesis, yet current methods often inadequately address the unique challenges presented by analysis of heterogeneous tissue samples. Here, we introduce Droplet Hi-C, which employs a commercial microfluidic device for high-throughput, single-cell chromatin conformation profiling in droplets. Using Droplet Hi-C, we can map the chromatin architecture at single-cell resolution from the mouse cortex and analyzed gene regulatory programs in major cortical cell types. Additionally, we can use this technique to detect copy number variations (CNVs), structural variations (SVs) and extrachromosomal DNA (ecDNA) in cancer cells, revealing clonal dynamics and other oncogenic events during treatment. Thus, Droplet Hi-C not only addresses critical gaps in chromatin analysis of heterogeneous tissues but also emerges as a versatile tool enhancing our understanding of gene regulation in health and disease.
Materials
Reagents
1×PBS (pH=7.4) (Thermo Fisher Scientific, Cat# 10010-23)
Sucrose (Sigma, Cat# S7903)
KCl (Invitrogen, Cat# AM9640G)
MgCl2 (Invitrogen, Cat# AM9530G)
Tris-HCl, pH 7.5 (ThermoFisher Scientific, Cat# 15567027)
DTT (Sigma, Cat# D9779)
37% formaldehyde (Sigma, Cat# 47608-250ML-F)
Glycine (Sigma, Cat# 50046-50G)
10% BSA (Sigma, Cat# A1595-50ML)
Tris-HCl, pH 8.0 (ThermoFisher Scientific, Cat# 15568025)
NaCl (Sigma, Cat# S5150)
Igepal CA630 (Sigma, Cat# I8896)
cOmplete EDTA-free Protease Inhibitor Cocktail (Roche, Cat# 05056489001)
SDS (Promega, Cat# V6551) Triton X-100 (Sigma, Cat# 93443)
10× Cutsmart buffer (NEB, Cat# M7204S)
DpnII (NEB, Cat# R0543L)
MboII (NEB, Cat# R0147M)
NlaIII (NEB, Cat# R0125L)
10× T4 DNA ligase buffer (NEB, Cat# B0202S)
20 mg/m BSA (NEB, Cat# B9000S)
T4 DNA ligase (NEB, Cat# M0202L)
7-AAD (Invitrogen, Cat# A1310)
Chromium Next GEM Single Cell ATAC Reagent Kits v2 (10x Genomics, Cat# 1000390)
Chromium Next GEM Chip H Single Cell Kit (10x Genomics, Cat# 1000161)
Single Index Kit N Set A (10x Genomics, Cat# 1000212)
SPRI beads (Beckman coulter, Cat# B23319)
Equipment
Benchtop centrifuge (Eppendorf)
T100 Thermal Cycler (Bio-Rad)
ThermoMixer (Eppendorf)
Swing bucket centrifuge (ThermoFisher)
Magnetic stands (10x Genomics)
Fragment analyzer/Tapestation (Agilent)
Micropipetts (0.5-20 μl, 10-200 μl, 100-1000 μl)
SH800 cell sorter (Sony)
RWD C100-Pro fluorescence cell counter (RWD)
Chromium iX or Controller (10x Genomics)
A. Nuclei preparation and fixation
A. Nuclei preparation and fixation
A-1 Nuclei preparation:
I, Cultured cells:
1. Disassociate the cells according to culture protocol.
2. Spin down cells at 500 g for 5 mins, resuspend the cells in culture medium.
3. Spin down again at 500 × g for 5 min, resuspend the cells in 1× PBS (pH=7.4).
4. Count the cells using cell counter.
II, Mouse brain tissues:
1. Prechill any tubes or tools, set the centrifuge to 4 °C.
2. Prepare Douncing Buffer: 0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris-HCl (pH 7.5), 1 mM DTT, 1× protease inhibitor, 0.5 U/μL RNaseOUT, 0.5 U/μL SUPERaseIn inhibitor.
3. Add 10 µL 10% Triton-X100 in to the douncer (1 mL), add 1 mL Douncing Buffer.
4. Transfer the mouse brain tissue dissection into douncer.
5. Use loose pestle 15-20 times gently followed by tight pestle for 15-20 times.
6. Filter with 30 µm Cell-Tric. Transfer to1.5 mL tubes. Spin down at 300 g for 10 min at 4°C.
7. Wash with 500 µL Douncing Buffer, and spin down again at 300 g for 10min.
8. Resuspend the nuclei in 1× PBS (pH=7.4).
9. Count the nuclei using cell counter.
A-2 Fixation:
1. Incubate 5-10 million cells/ nuclei in 10 mL 1% formaldehyde, which is diluted with 1× PBS (pH=7.4), at room temperature for 10 min.
2. Quench formaldehyde in 200 mM glycine and incubate at room temperature for 5 min.
3. Spin down cells/ nuclei at 1,000 g for 5 min at 4°C, then resuspend using 1% BSA in 1× PBS (pH=7.4) to wash twice.
4. Count the cells/ nuclei using cell counter.
5 Aliquot one million cells/ nuclei per tube.
6. Spin down cells/ nuclei at 1,000 g for 5 min at 4°C. remove the supernatant and flash freeze the pellet in liquid nitrogen.
7. Store the crosslinked cells/ nuclei at -80°C.
B. In situ Hi-C treatment and barcoding
B. In situ Hi-C treatment and barcoding
B-1 In situ Hi-C treatment:
1. Resuspend frozen crosslinked cells/ nuclei with pre-cold 300 μL lysis buffer (10 mM Tris-HCl, pH 8.0, 10 mM NaCl, 0.2% Igepal CA630, 1×protease inhibitor) on ice for 45 min.
2. Spin down at 1,000 g for 5 min at 4°C to collect nuclei, and wash once with 200 μL lysis buffer.
3. Resuspend the nuclei with 50 μL 0.5% SDS and incubate at 62°C for 10 min on Thermomixer.
4. Quench SDS by adding 145 μL of nuclease-free H2O and 25 μL 10% Triton X-100, incubate at 37°C for 15 min on Thermomixer at 300 rpm.
5. Add 27 μL of 10× CutSmart Buffer and three restriction enzymes, including 50 U DpnII, 62.5U MboII and 7.5U NlaIII, and then incubate at 37°C for 90 min on Thermomixer at 550 rpm.
6. Heat inactivation restriction enzymes at 65°C for 20 min and then cool down to room temperature.
7. Collect nuclei by centrifugation at 1000 g for 5 min at 4°C and wash once with 200 μL ligation buffer (100 μL 10× T4 DNA ligase buffer, 5 μL 20 mg/mL BSA, 865 μL H2O).
8. Add 200 μL ligation buffer and 20 μL T4 DNA ligase and incubate at 37°C for 40 min on Thermomixer at 300 rpm.
B-2 Nuclei sorting (Optional):
1. Spin down the nuclei at 1,000 g for 5 min at 4°C and then resuspend in 1 mL sorting buffer (1% BSA in PBS (pH=7.4)), add 1 μL 1,000× 7-AAD.
2. Sort nuclei by fluorescence-activated nuclei sorting with an SH800 cell sorter (Sony) for the isolation of single nuclei.
3. Collect nuclei in collection buffer (5% BSA in PBS (pH=7.4)) at 5°C.
B-3 Nuclei tagmentation and barcoding
1. Spin down for 5 min at 1,000 g and 4 °C to collect nuclei.
2. Wash the nuclei once with 1x Nuclei Buffer (diluted from 20× Nuclei Buffer (10x Genomics, PN-2000207)).
3. Resuspend nuclei in 10 μL 1x Nuclei Buffer and then do nuclei counting with DAPI staining.
4. Aliquot 3,000-15,000 nuclei to PCR tubes according to the targeted nuclei recovery number.
5. Prepare tagmentation reaction following user guide of Chromium Next GEM Single Cell ATAC Reagent Kits v1.1 or Chromium Next GEM Single Cell ATAC Reagent Kits v2.
6. Incubate tagmentation system at 37 °C for 60 min.
7. Perform GEM Generation & Barcoding following user guide of Chromium Next GEM Single Cell ATAC Reagent Kits v1.1 or Chromium Next GEM Single Cell ATAC Reagent Kits v2.
C. Single-cell Hi-C sequencing Library construction
C. Single-cell Hi-C sequencing Library construction
1. The protocol of Post GEM Incubation Cleanup is the same as described in user guide of Chromium Next GEM Single Cell ATAC Reagent Kits v1.1 or Chromium Next GEM Single Cell ATAC Reagent Kits v2.
2. The protocol of Library Construction is almost the same as described in user guide of Chromium Next GEM Single Cell ATAC Reagent Kits v1.1 or Chromium Next GEM Single Cell ATAC Reagent Kits v2. The modified step here is that extending index PCR elongation time from 20s to 1 min. The double-sided size selection was changed to 1.14x SPRIselect to only remove small fragments.
D. Library sequencing
D. Library sequencing
1. All sequencing experiments were performed with an Illumina NextSeq 2000 or NovaSeq 6000 sequencer. The 200 cycles kits are used, with cycle number set as 100+8+16+100 (Read1 100 cycles; Index1 8 cycles; Index2 16 cycles; Read2 100 cycles).