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| Status |
Public on Jan 02, 2023 |
| Title |
HiC_ESC_WT_Rep2 |
| Sample type |
SRA |
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| Source name |
Embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: Embryonic stem cells
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| Treatment protocol |
2x10^6 cells were dissociated in a single-cell-suspension using pre-warmed Trypsin-EDTA (0.05%) and incubated for 5-10 min at 37°C. Trypsin was blocked by adding 10% FCS/DPBS and cells were centrifuged for 5 min at 1100 rpm. Cell pellet was resuspended in a 2% PFA (in 10%FCS/DPBS) fixation solution and incubated at room temperature for 10 min while tumbling. The reaction was quenced on ice by adding Glycine (final concentration 125mM) and cells were collected by centrifugation at 400 g for 8 min at 4°C.
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| Growth protocol |
Embryonic stem cells (ESC) cells were grown on gelatin/MEF-coated cell culture dishes containing ESC medium (Knockout DMEM (Thermo, #10829018, 15% fetal bovine serum (PAN, #P30-2602), 1X GlutaMAX supplement (Thermo, #35050-038), 1X non-essential amino acids (Thermo, #11140-035), 100 µM 2-mercaptoethanol (Thermo, #21985023), 1X Penicillin/Streptomycin (Thermo, #15140122) and 1X leukemia inhibitory factor (LIF) (selfmade). Splitting was carried out every three days by rinsing the cells once with DPBS (Thermo, #14190144) before detaching the cells using TryplE (Gibco, #12604013). Before sample collection, ESCs were passaged at least one passage without MEFs to dilute out feeder cells. During this time, the cells were cultured in ESC medium containing 2X LIF. Trophoblast stem cells (TSCs) cells were grown on MEF-coated cell culture dishes containing TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149). Splitting was carried out every five to seven days by rinsing the cells once with DPBS (Thermo, #14190144) before detaching the cells using Trypsin-EDTA (0.05%)(Thermo, #25300054). TSCs were passaged in clumps. Before sample collection, TSCs were passaged at least one passage without MEFs to dilute out feeder cells. During this time, the cells were cultured in MEF-conditioned TSC medium (70 % MEF-conditioned medium, 30 % TSC medium, +FGF4 (37.5 ng/ml), Heparin (1.5 µg/ml)).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
To extract the nuclei, cells were incubated on ice for 10 min with ice-cold Lysis Buffer (50mM Tris HCl pH 7.5, 150 mM NaCl, 5 mM 788 EDTA, 0.5% NP-40, 1.15% Triton X-100, 25X Protease Inhibitor in Milli-Q Water). Extracted nuclei were centrifuged at 750g for 5 min at 4°C, two times washed with 1X DPBS, snap frozen and stored at -80°C.
Nuclei were first permeabilized using 0.5% SDS at 62°C for 10 min and later, chromatin was digested with DpnII (NEB, #R0543) for 90 min at 37°C with gentle rotation. The overhangs generated by digestion were filled and marked with biotin-14-dATP (Thermo, #19524016) by a 90 min incubation at 37°C with gentle rotation. After, DNA fragments were ligated for 4 hours at 20°C with gentle rotation using T4 DNA Ligase (Thermo, #M0202M). Chromatin was then reverse-crosslinked, precipitated and sheared with Covaris S220 (2 cycles, each 50sec long; 10% duty; 4 intensity; 200 cycles/burst). The biotin-marked-DNA shared fragments were pulled down using Dynabeads MyOne Streptavidin T1 beads (Thermo, #65601), purified and further processed for Illumina sequencing with NEBNext Ultra II Library Prep Kit for Illumina according to the kit guidelines (NEBNext End Prep, Adaptor Ligation, PCR enrichment of Adaptor-Ligated DNA using NEBNext Multiplex Oligos for Illumina). Clean up and size selection were performed with AMPure beads.
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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 |
Raw reads were subjected to adapter and quality trimming with cutadapt (version 2.4; parameters: --quality-cutoff 20 --overlap 5 --minimum-length 25 --adapter AGATCGGAAGAGC -A AGATCGGAAGAGC).
Mates were separately aligned to the mouse genome (mm10) using bwa with the ‘mem’ command (version 0.7.17; parameters: -A 1 -B 4 -E 50 -L 0).
Hi-C matrices for each replicate were built using HiCExplorer with the ‘hicBuildMatrix’ command (version 3.6; parameters: --binSize 5000 --restrictionSequence GATC --danglingSequence GATC --minMappingQuality 30).
TSC and ESC replicates were merged respectively using ‘hicSumMatrices’. TSC and ESC matrices were normalized together using ‘hicNormalize’ (parameters: --smallest) and corrected using ‘hicCorrectMatrix’ (parameters: --correctionMethod KR).
For whole-chromosome representation and compartment analysis bins of matrices at 5 kb resolution were merged into 100 kb bins using ‘hicMergeMatrixBins’ (parameters: -nb 20).
Assembly: mm10
Supplementary files format and content: Normalized and KR-corrected matrices of 5 kb resolution in h5 format.
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| Submission date |
May 31, 2022 |
| Last update date |
Jan 02, 2023 |
| Contact name |
Sara Hetzel |
| E-mail(s) |
hetzel@molgen.mpg.de
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| Organization name |
Max Planck Institute for Molecular Genetics
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| Department |
Genome Regulation
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| Lab |
Meissner Lab
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| Street address |
Ihnestraße 63
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| City |
Berlin |
| ZIP/Postal code |
14195 |
| Country |
Germany |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE166362 |
Dynamic antagonism between key repressive pathways maintains the placental epigenome |
| GSE205167 |
Dynamic antagonism between key repressive pathways maintains the placental epigenome (Hi-C) |
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| Relations |
| BioSample |
SAMN28777727 |
| SRA |
SRX15509860 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6206662_HiC_ESC_WT_Rep2_5kb_mm10.h5 |
2.5 Gb |
(ftp)(http) |
H5 |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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