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| Status |
Public on Jan 23, 2017 |
| Title |
ESC_H3K27me3_ChIPseq_Rep1 |
| Sample type |
SRA |
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| Source name |
embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
strain: 129/Sv
genoptype: wild type
chip antibody: H3K27me3 (Millipore; 170622)
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| Growth protocol |
mES cells were maintained on a feeder layer in mESC growth medium: Dulbecco’s modified Eagle’s medium (Gibco; 11965) containing 2 mM L-glutamine (Sigma), supplemented with 15% (v/v) FBS (Atlanta Biologicals; S12450), 0.1 mM nonessential amino acids (Sigma; M7145), 1 mM sodium pyruvate (Invitrogen; 11360070), 1000 units/mL mLIF (Millipore; ESG1106), penicillin and streptomycin (Invitrogen; 15140), and 0.1 mM b-mercaptoethanol (Sigma). The cells were passaged twice to eliminate feeder cells before being used in experiments.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
mESCs were passaged twice without a feeder layer and then grown in geletin-coated plates to ~70 to 80% confluence. The cells were cross-linked using 1% paraformaldehyde at room temperature for 10 min., followed by quenching in 125 mM glycine for 5 min. at 4°C. The crosslinked cells were collected, and the nuclei were released by gentle pipetting three times in lysis buffer [10 mM Tris-HCl pH 7.5, 2 mM MgCl2, 3 mM CaCl2, 0.5% NP-40, 10% glycerol, 1 mM DTT, 1x protease inhibitor cocktail (Roche)]. The nuclei were collected by gentle centrifugation and resuspended in sonication buffer [1x PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1x protease inhibitor cocktail (Roche)]. The nuclei were then incubated in sonication buffer on ice for 10 min., and sonicated at 4°C using a Biorupter (Diagenode) high setting, three cycles of 5 min. sonication (30 seconds on/30 seconds off) with 5 min. intervals) to generate genomic DNA fragments of 200 - 500 bp in length. The sonicated chromatin was clarified by centrifugation and pre-cleared with agarose beads.
Aliquots of the pre-cleared chromatin were immunoprecipitated with various antibodies at 4°C overnight, followed by collection of the immunoprecipitates using protein A (Millipore) or G (Invitrogen) agarose beads (protein A beads for H3K4me3, H3K27me3, and PARP-1 antibodies; protein G beads for Sox2 and Oct4 antibodies). The beads were collected by gentle centrifugation and washed in low salt wash buffer (20 mM Tris-HCl pH 7.9, 2 mM EDTA, 125 mM NaCl, 0.05% SDS, 1% Triton X-100, 1x protease inhibitor cocktail), high salt wash buffer (low salt wash buffer containing 500 mM NaCl), and LiCl wash buffer (10 mM Tris-HCl pH 7.9, 1 mM EDTA, 250 mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1x protease inhibitor cocktail). The immunoprecipitated genomic DNA was eluted and the crosslinks were reversed by incubation in elution buffer (100 mM NaHCO3, 1% SDS) at 65°C overnight. The genomic DNA was then deproteinized by digestion with proteinase K and extraction with phenol:chloroform:isoamyl alcohol, followed by precipitation with ethanol. Approximately 50 ng of ChIPed DNA (quantified using a NanoDrop) was used to prepare each ChIP-seq library. The genomic DNA fragments were end-polished, dA-tailed, and ligated to Y-adaptors containing barcode sequences. After agarose gel-based size selection and purification, the DNA was amplified for 13 - 15 cycles by PCR using Phusion high-fidelity DNA polymerase (NEB). The final ChIP-seq libraries were subjected to quality control assessment using a Bioanalyzer (Agilent), followed by 50 bp sequencing using an Illumina HiSeq 2000 Sequencing System.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
ChIP-seq reads were subjected to quality-control using FastQC
trimmed to remove adapter sequences
and aligned to the mouse reference genome (mm9) using Bowtie 0.12.7 allowing for one mismatch during alignment.
The data were then converted to wiggle (WIG) file format using PeakRanger
Genome_build: mm9
Supplementary_files_format_and_content: wiggle file for visualization
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| Submission date |
Oct 16, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
W. Lee Kraus |
| E-mail(s) |
lee.kraus@utsouthwestern.edu
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| Organization name |
UT Southwestern Medical Center
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| Street address |
5323 Harry Hines Blvd.
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| City |
Dallas |
| State/province |
TX |
| ZIP/Postal code |
75390-8511 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE74112 |
Genome-wide maps of histone marks, Sox2, Oct4 and PARP-1 in mES cells. |
| GSE81168 |
Regulation of gene transcription and transcription factor chromatin binding by PARP-1 in mouse embryonic stem cells |
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| Relations |
| BioSample |
SAMN04192258 |
| SRA |
SRX1342331 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1910634_H3K27me3_WT.wig.gz |
45.4 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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