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| Status |
Public on Jul 28, 2023 |
| Title |
Context-dependent perturbations in chromatin folding and the transcriptome by cohesin and related factors [ChIP-seq] |
| Organism |
Homo sapiens |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
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| Summary |
Cohesin plays vital roles in chromatin folding and gene expression regulation, cooperating with such factors as cohesin loaders, unloaders, and the insulation factor CTCF. Although models of regulation have been proposed (e.g., loop extrusion), how cohesin and related factors collectively or individually regulate the hierarchical chromatin structure and gene expression remains unclear. We have depleted cohesin and related factors and then conducted a comprehensive evaluation of the resulting 3D genome, transcriptome and epigenome data. We observed substantial variation in depletion effects among factors at topologically associating domain (TAD) boundaries and on interTAD interactions, which were related to epigenomic status.
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| Overall design |
Chromatin preparation for ChIP was performed as described (Izumi et al., Nat Genet. 2015, 10.1038/ng.3229). In brief, ~8 × 10^6 RPE cells were crosslinked with 1% formaldehyde for 10 min at room temperature, followed by an additional 5 min with glycine in PBS added at a final concentration of 125 mM. Fixed cells were lysed in LB1 (50 mM HEPES-KOH, pH 7.4; 140 mM NaCl; 1 mM EDTA; 10% glycerol; 0.5% NP-40; 0.25% Triton X-100; 10 mM dithiothreitol; 1 mM PMSF) on ice. The lysate was washed sequentially with LB2 (20 mM Tris-HCl, pH 8.0; 200 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 1 mM PMSF) and LB3 (20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 1% Triton X-100; 0.1% sodium deoxycholate; 0.1% SDS; 1× cOmplete protease inhibitor cocktail [Roche]) on ice. The lysate was resuspended in LB3 and sonicated using Branson Sonifier 250D (Branson) for chromatin shearing (12 sec with amplitude setting at 17% of the maximum amplitude, six times). In addition, lysate containing fragmented chromatin was also prepared from ~2 × 10^6 mouse cells (C2C12) with the same procedures. Human cell lysate and mouse cell lysate (as a spike-in internal control) were combined (~4:1 ratio) and incubated with protein A or G Dynabeads (Thermo Fisher Scientific) conjugated with the relevant antibodies for 14 h at 4℃. The beads were then washed five times with cold RIPA wash buffer (50 mM HEPES-KOH, pH 7.4; 500 mM LiCl; 1 mM EDTA; 0.5% sodium deoxycholate; 1% NP-40) and once with cold TE50 (50 mM Tris-HCl, pH 8.0; 10 mM EDTA). Material captured on the beads was eluted with TE50 containing 1% SDS. The eluted material and input were incubated for 6 h at 65℃ to reverse crosslinks and were treated with 100 ng RNaseA (Roche) for 1 h at 50℃, followed by treatment with 100 ng Proteinase K (Merck) overnight at 50℃. The input and ChIP DNA were then purified with a PCR purification kit (Qiagen). DNA from the ChIP and input fractions was end-repaired, ligated to sequencing adaptors, amplified and size-selected using NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) and Agencourt AMPure XP (Beckman Coulter). DNA was then sequenced to generate single-end 65-bp reads using the Illumina HiSeq-2500 system.
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| Web link |
https://www.nature.com/articles/s41467-023-41316-4
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| Contributor(s) |
Nakato R, Sakata T, Bando M, Shirahige K |
| Citation(s) |
37726281 |
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| Submission date |
Feb 14, 2022 |
| Last update date |
Oct 03, 2023 |
| Contact name |
Ryuichiro Nakato |
| E-mail(s) |
rnakato@iqb.u-tokyo.ac.jp
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| Phone |
+81-3-5841-1471
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| Organization name |
The University of Tokyo
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| Department |
Institute for Quantitative Biosciences
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| Lab |
Laboratory of Computational Genomics
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| Street address |
1-1-1 Yayoi
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| City |
Bunkyo-ku |
| State/province |
Tokyo |
| ZIP/Postal code |
113-0032 |
| Country |
Japan |
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| Platforms (2) |
| GPL16791 |
Illumina HiSeq 2500 (Homo sapiens) |
| GPL30173 |
NextSeq 2000 (Homo sapiens) |
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| Samples (61)
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| This SubSeries is part of SuperSeries: |
| GSE196450 |
Context-dependent perturbations in chromatin folding and the transcriptome by cohesin and related factors |
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| Relations |
| BioProject |
PRJNA806974 |
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