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| Status |
Public on Mar 30, 2022 |
| Title |
H3K27Ac_ESC_dTAG_24h_rep3 |
| Sample type |
SRA |
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| Source name |
mESC
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| Organism |
Mus musculus |
| Characteristics |
cell line: V6.5
treatment: dTAG
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| Treatment protocol |
For degradation of TRIM28, 500 nM of dTAG-13 (or 0.05 % DMSO) was mixed with mESC medium and added to the cells 24h before samples were collected.
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| Growth protocol |
During expansions, cells were cultured on irradiated primary mouse embryonic fibroblasts (MEFs) under serum/LIF conditions: knockout DMEM containing 15 % fetal bovine serum, 50 µg/ml penicillin/streptomycin, 2 mM L-Glutamine, 100 µM non-essential amino acids, 80 µM ß-mercaptoethanol and 1000 U/ml LIF. Before starting ChIP-Seq experiments, mESCs were depleted from MEFs by incubating them on gelatin coated cell culture plates for 45 min in +37 C allowing MEFs to attach while mESCs remain in suspension. MEF depletion was performed two times after which mESCs were seeded on gelatin coated plates and maintained in serum/LIF conditions with 2000 U/ml LIF.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were detached with TrypLE, washed once with PBS, fixed with 1 % formaldehyde for 10 minutes in room temperature followed by quenching with 125 mM glycine. For each sample, three million mESCs and 750 000 S2 cells were lysed in LB1 (50 mM HEPES-KOH, 140 nM NaCl, 1 mM EDTA, 10 % glycerol, 0.5 % Igepal ca-630 and 0.25 % Triton X-100, 5 mM Na-butyrate and 1x protease inhibitor cocktail) and collected by sentrifugation. Lysis was continued in LB2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 5 mM Na-butarate and 1 x protease inhibitor cocktail) followed by sentrifugation, nuclei were lysed in LB3 (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1 % Na-deoxycholate, 0.5 % N-Lauroylsarcosine, 5 mM Na-butyrate and 1x protease inhibitor cocktail) and chromatin fragmented with Bioruptor NextGen for 35 cycles (high-setting). Lysates were clarified and protein-DNA complexes were isolated with ab4729 (Abcam) antibody for H3K27Ac and ab8898 (Abcam) antibody for H3K9me3 and Protein A Dynabeads (Invitrogen). Beads were washed 7 times with RIPA buffer (50 mM HEPES-KOH pH7.5, 1 mM EDTA, 1 % Igepal CA-630, 0.7 % Na-deoxycholate, 500 mM LiCl, 5 mM Na-butyrate and 1 x protease inhibitor cocktail), once with TE buffer (10 mM Tris-HCl pH8.0, 1 mM EDTA and 50 mM NaCl) and eluted from beads with Elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA and 1% SDS). Samples were decrosslinked for 16 h in +65 C with 550 mM NaCl and proteinase K, treated with RNAseA and DNA was extracted with Phenol:Chloroform:Isoamylalcohol followed by chloroform extraction and precipitation with 30 µg glycogen, 0.3 M Na-acetate and ethanol.
10 ng of DNA was used to prepare sequencing libraries with KAPA HyperPrep Kit (Roche) according to manufacturer's instructions.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
ChIP sequencing of mESCs
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| Data processing |
Raw reads of treatment and input samples were subjected to adapter and quality trimming with cutadapt (version 2.4; parameters: --nextseq-trim 20 --overlap 5 --minimum-length 25 --adapter AGATCGGAAGAGC -A AGATCGGAAGAGC). Reads were aligned separately to the mouse genome (mm10) and to the fly genome (D. Melanogaster, dm6) using bwa with the 'mem' command (version 0.7.17, default parameters). A sorted BAM file was obtained and indexed using samtools with the 'sort' and 'index' commands (version 1.10). Duplicate reads were identified and removed using gatk (version 4.1.4.1) with the 'MarkDuplicates' command and default parameters. Technical replicates of treatment and input samples were merged respectively using samtools 'merge'.
Peaks were called with reads aligning to the mouse genome only using MACS2 'callpeak' (version 2.1.2; parameters --bdg --SPMR) using the input samples as control samples. For H3K9me3 only, the '--broad' option was used. Genome-wide coverage tracks for single and merged replicates normalized by library size were computed using bamCoverage (version: 3.4.3; parameters: --normalizeUsing CPM --extendReads) and in addition normalized by the spike-in factor obtained from the reads aligning to the drosophila genome.
Genome_build: mm10
Supplementary_files_format_and_content: bigWig coverage files
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| Submission date |
Oct 13, 2020 |
| Last update date |
Mar 30, 2022 |
| 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 (1) |
| GSE159468 |
Hijacking of transcriptional condensates by endogenous retroviruses |
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| Relations |
| BioSample |
SAMN16428927 |
| SRA |
SRX9286204 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4830134_H3K27Ac_ESC_dTAG_24h_rep3.bw |
214.9 Mb |
(ftp)(http) |
BW |
| GSM4830134_H3K27Ac_ESC_dTAG_24h_rep3_narrowPeak.bed.gz |
1.1 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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