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| Status |
Public on Oct 09, 2024 |
| Title |
HiC_ES_dKO_SRF_Cre_CTCF_dTAG13 |
| Sample type |
SRA |
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| Source name |
Srf_flox1
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| Organism |
Mus musculus |
| Characteristics |
cell line: Srf_flox1
cell type: embryonic stem cells
genotype: SRF/CTCF dKO
treatment: untreated
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
HiC was performed using a total of 5x105 cells per sample. Cells were treated and collected as follows. For non-differentiated ESCs, 2x106 Srf-flox1 cells encoding endogenous CTCF-FKBP were deprived of feeder cells and transfected with 25 μg GFP (Addgene, #11150) or 25 μg GFP-Cre plasmid (Addgene, #13776) using Lipofectamine 2000. 24 hours after the transfection, the medium was refreshed and supplemented with 250 nM dTAG13 or the matching volume of DMSO. After 24 hours of incubation with dTAG13 or DMSO, 5x105 of GFP-positive WT (GFP + DMSO), SRF KO (GFP-Cre + DMSO), CTCF KO (GFP + dTAG13), or SRF/CTCF dKO (GFP-Cre + dTAG13) cells were sorted using FACS Aria II. For dTAG13-induced SRF KO ESCs, 1x106 F1G4 cells encoding endogenous SRF-FKBP were treated with 250 nM dTAG13 for 24 hours, and 5x105 cells were collected using FACS Aria II. For CM cells, Srf-flex1 cells carrying H2B-mCherry (WT) or H2B-Venus-T2A-Cre (KO) were trypsinized at day 4 of cardiac mesoderm differentiation, and 5x105 cells expressing mCherry or Venus were sorted on a FACS Aria II. Sorted cells were crosslinked using Low Input crosslinking protocol provided by the Arima-HiC Kit user guide (A 160134 v01) and frozen in liquid nitrogen.
Proximally-ligated DNA was generated using Arima-HiC Kit (A510008) following the manufacturer’s protocol. HiC library was generated using KAPA Hyper Prep Kit (Roche, 07962312001) and KAPA single-indexed adapters (Roche, 08005702001) according to Arima’s Library preparation user guide. Covaris S220 was used for DNA shearing. The libraries were quantified using the Qubit DNA HS assay and the library size was validated using DNA HS Bioanalyzer chips. Paired-end sequencing was performed on the HiSeq 4000 with 2x 75 bp read length or on the NovaSeq 6000 with 2x 100 bp read length.
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| Library strategy |
Hi-C |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
The Hi-C reads were mapped using HiC-Pro (3.1.0) with the following bowtie2 arguments: --very-sensitive -L 30 --score-min L,-0.6,-0.2 --end-to-end --reorder (global); --very-sensitive -L 20 --score-min L,-0.6,-0.2 --end-to-end --reorder (local). Genome fragment file for the HiC-Pro was generated for the following ligation sites: GATCGATC, GAATGATC, GATTGATC, GACTGATC, GAGTGATC, GATCAATC, GATCATTC, GATCACTC, GATCAGTC, GAATAATC, GAATATTC, GAATACTC, GAATAGTC, GATTAATC, GATTATTC, GATTACTC, GATTAGTC, GACTAATC, GACTATTC, GACTACTC, GACTAGTC, GAGTAATC, GAGTATTC, GAGTACTC, GAGTAGTC and using MIN_INSERT_SIZE = 100, MAX_INSERT_SIZE = 900. Independent replicates were mapped individually and valid pairs (allValidPairs file generated by HiC-Pro) were concatenated to generate contact matrices. Hi-C contact matrices in h5 format at 10 kb resolution were generated using hicBuildMartix tool from HiCExplorer (3.7.2) [Ramirez, 2018] with the following arguments: --minMappingQuality 10 --skipDuplicationCheck -rs ResSites_combined.bed -seq GATC GAATC GATTC GACTC GAGTC --danglingSequence GATC AAT ATT ACT AGT -bs 10000. hicBuildMatrix input sam files containing the mapped reads were generated by converting the allValidPairs into the sam format using custom scripts. The file containing all restriction cut sites (ResSites_combined.bed) was created using hicFindRestSite tool with the argument --searchPattern GATC, followed by the argument --searchPattern GA.TC and concatenation of the output bed files. For independent replicates, the h5 format matrices were merged using hicSumMatrices tool from HiCExplorer. The merged matrices were corrected using hicCorrectMatrix tool with the Knight-Ruiz (KR) matrix balancing algorithm (--correctionMethod KR). Hi-C contact matrices in hic format for Juicebox and Juicer tools analyses were created from a list of all valid pairs (allValidPairs file generated by HiC-Pro; for independent replicates, allValidPairs files were concatenated) using hicpro2juicebox.sh script from the HiC-Pro Utilities with the following argument: -j juicer_tools.2.20.00.jar.
genome build/assembly: mm10
processed data files format and content: Contact matrices in h5 (at 20kb resolution, corrected using Knight and Ruiz (KR) balancing algorithm) and hic formats. For normalization, merging or other processing steps, HiCExplorer (h5) or Juicer tools (hic) can be used.
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| Submission date |
Feb 13, 2024 |
| Last update date |
Oct 09, 2024 |
| Contact name |
Pavel Tsaytler |
| Organization name |
MPI Molecular Genetics
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| Street address |
Ihnestrasse 63-73
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| City |
Berlin |
| ZIP/Postal code |
14195 |
| Country |
Germany |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE255733 |
SRF promotes long-range chromatin loop formation and stem cell pluripotency |
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| Relations |
| BioSample |
SAMN39942349 |
| SRA |
SRX23619667 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8078720_HiC_ES_dKO_SRF_Cre_CTCF_dTAG13.hic |
1.0 Gb |
(ftp)(http) |
HIC |
| GSM8078720_HiC_ES_dKO_SRF_Cre_CTCF_dTAG13_20kb_KR-corrected.h5 |
478.4 Mb |
(ftp)(http) |
H5 |
SRA Run Selector |
| Raw data are available in SRA |
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