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Sample GSM820727 Query DataSets for GSM820727
Status Public on Nov 30, 2011
Title EedKO-CBX7
Sample type SRA
Source name Embryonic stem cells WT
Organism Mus musculus
Characteristics cell type: embryonic stem cells
strain: C57BL/6
genotype/variation: Eed -/-
chip antibody: CBX7
chip antibody vendor: Abcam
chip antibody catalog #: ab21873
Treatment protocol Cells (1-2x108 cells) were fixed in PBS with 2 µM EGS (21565, thermofisher) for 1 h at RT. After washing cells were fixed again in ChIP fix buffer (1% formaldehyde, 5 µM EGTA, 10 µM EDTA, 1 mM NaCl and 0.5 mM HEPES in PBS) for 10 min at RT. Fixation was stopped by Glycine to a final concentration of 125 mM. Cell extracts were lysed (1% SDS, 10 mM EDTA pH 8.0, 50 mM Tris-HCl pH 8.1) with complete protease inhibitors (Roche) and sonication was performed using a BioRupter sonicator (Diagenode) to obtain an average DNA fragment size of 300 bp. Chromatin was diluted with 1% Triton X-100, 2 mM EDTA pH 8.0, 150 mM NaCl, 20 mM Tris-HCl pH 8.1 containing complete protease inhibitors. Protein G Sepharose and rProtein A Sepharose (17-0618-01 and 17-1279-01, respectively, GE Healthcare) were blocked for 1 h at 4 oC with 1 mg/ml BSA and 1 mg/ml yeast tRNA (R8759-500UN, Sigma). Chromatin was pre-cleared with blocked beads for 1 h at 4 oC. 150 µg of chromatin were incubated with Ring1B antibodie(Atsuta, T. et al. Production of monoclonal antibodies against mammalian Ring1B proteins. Hybridoma 20, 43-6 (2001)) ON at 4 oC with rotation. Protein-antibody complexes were pulled down by adding beads to the solution for 2 h. Complexes were washed 4 times with 1% SDS, 1% triton X-100, 2 mM EDTA pH 8.0, 150 mM NaCl and 200 mM Tris-HCl pH 8.0, followed by 1 washes in 0.1% SDS, 1% triton X-100, 500 mM NaCl and 20 mM Tris-HCl pH 8.1. The last wash was in TE. Samples were treated with RNase A and Proteinase K and reverse crosslinked ON. DNA was then purified using a PureLink PCR micro column (Invitrogen).
Extracted molecule genomic DNA
Extraction protocol ChIP DNA was end-repaired, A-tailed and adapter-ligated using a ChIP-seq DNA Sample prep Kit (Illumina). A Qiagen PCR purification step was performed following each enzymatic reaction. The adapter-ligated material was then PCR amplified, using 18 cycles of PCR before size selection of 200-500 bp fragments on an agarose gel. The library was then extracted using a Qiaquick gel extraction kit and checked for concentration and integrity.
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 2000
Data processing Pooled replicates were aligned using BOWTIE (Please cite: Langmead B, Trapnell C, Pop M, Salzberg SL. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10:R25) using parameter -m 1 --best, visualized using GBrowser
Peaks: Peak detection was performed with the Model-based Analysis of ChIP-Seq (MACS) algorithm ( using an FDR of <2 and number of tags >100
genome build: mm9
Submission date Oct 21, 2011
Last update date May 15, 2019
Contact name Stephen Taylor
Phone +44 1865 222640
Organization name CBRG
Street address Headington
City Oxford
ZIP/Postal code OX3 9DS
Country United Kingdom
Platform ID GPL13112
Series (1)
GSE23716 H3K27me3 is not required for recruitment of Polycomb repressor complex 1 to target loci in mouse embryonic stem cells
SRA SRX101808
BioSample SAMN00740099

Supplementary file Size Download File type/resource
GSM820727_Eed_KO_CBX7.bam 3.7 Gb (ftp)(http) BAM
SRA Run SelectorHelp
Processed data provided as supplementary file
Raw data are available in SRA

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