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| Status |
Public on Mar 24, 2021 |
| Title |
ESC_Ctrl_H3K4me1_ChIP_rep1 |
| Sample type |
SRA |
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| Source name |
mouse ES cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: ES cells
cell line: E14
antibody: H3K4me1 (Cell Signaling, 5326)
treatment: Control
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| Treatment protocol |
A-485 (10uM) or JQ1(0.5mM) were treated for 60 min if not indicated. For control samples, 0.1 % DMSO was added.
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| Growth protocol |
E14Tg2A Rosa26-Cre Oct4-IRES-Puro mouse ESCs were used for all experiments 11932748. ESCs were cultured in a custom-made N2B27 medium consisting of 1:1 mix of DMEM/F12 and Neurobasal medium, without L-arginine and L-lysine (C.C.Pro GmbH, Oberdorla). The media was supplemented with L-arginine, L-lysine, 1000 units/ml leukemia inhibitory factor (LIF) (Merck Millipore), 1µM PD0325901, 3µM CT-99021 (custom-made by ABCR), 100µM 2-mercaptoethanol, 150µM sodium pyruvate, 0.5x B27 supplement (Thermo Fisher Scientific), as well as with 0.5x N2 supplement (made in house; or from Thermo Fisher Scientific).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were cross-linked for 10 min at room temperature with 1% formaldehyde solution with gentle agitation and quenched with 0.125 M glycine. Cross-linked cells were washed once with PBS, added swelling buffer (10 mM Tris-HCl pH8.0, 1.5 mM MgCl2, 10 mM NaCl, 0.5% NP-40) and incubated on ice for 10 minutes. After centrifugation, pellets were suspended with RIPA buffer (10 mM Tris-HCl pH8.0, 1 mM EDTA, 140 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% Sodium Deoxycholate) containing proteinase inhibitor cocktail (Roche), and then were sonicated by using a BioRuptor sonicator (Diagenode). After centrifugation, solubilized chromatin was recovered and incubated with 5 µg of antibody bound to protein A/G magnetic beads (Pierce). After overnight incubation at 4ºC, the magnetic beads were washed with RIPA buffer twice, RIPA high salt buffer (10 mM Tris-HCl pH8.0, 1 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% Sodium Deoxycholate), LiCl buffer (10 mM Tris-HCl pH8.0, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% Sodium Deoxycholate) and TE buffer. The bound materials were eluted with elution buffer (10 mM Tris-HCl pH8.0, 5 mM EDTA, 300 mM NaCl, 0.1% SDS) overnight at 65ºC and treated with RNaseA for 30 min for 37°C and further incubation with Proteinase K for 1 hour at 37ºC. Then DNA was purified with a PCR purification kit (Qiagen).
ChIP-seq libraries were prepared by using the NEBNext Ultra II DNA prep kit (NEB E7645) following the manufacturer's instruction and sequenced on a Next-Seq 500 Sequencer (Illumina).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
processed data file: ChIP.ESC_Ctrl_H3K4me1_TC0.n2.smooth.bw
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| Data processing |
Read alignment was performed using BWA with default parameters. Low mapping quality read (MAPQ 10) and duplicated reads were removed using samtools. The DAC Blacklisted Regions were also omitted from the bam file using bedtools. Peaks were called using epic with the following parameters: --fragment-size 200 --gaps-allowed 5.
Genome_build: mm10
Supplementary_files_format_and_content: bigWig
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| Submission date |
Sep 25, 2020 |
| Last update date |
Mar 24, 2021 |
| Contact name |
Chunarum Choudhary |
| E-mail(s) |
chuna.choudhary@cpr.ku.dk
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| Organization name |
University of Copenhagen
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| Department |
Novo Nordisk Foundation Center for Protein Research
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| Lab |
Choudhary Group
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| Street address |
Blegdamsvej 3B
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| City |
Copenhagen N |
| State/province |
--- Select a state --- |
| ZIP/Postal code |
2200 |
| Country |
Denmark |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE146324 |
Enhancers are Activated by p300/CBP Activity-Dependent PIC Assembly, RNAPII Recruitment and Pause Release [ChIP-Seq] |
| GSE146328 |
Enhancers are Activated by p300/CBP Activity-Dependent PIC Assembly, RNAPII Recruitment and Pause Release |
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| Relations |
| BioSample |
SAMN16267971 |
| SRA |
SRX9195269 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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