GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
Sample GSM585231 Query DataSets for GSM585231
Status Public on Nov 30, 2011
Title EedKO-Ring1B replicate 2
Sample type SRA
Source name Embryonic stem cells knockout for Eed
Organism Mus musculus
Characteristics strain: C57BL/6
cell type: embryonic stem cells
genotype/variation: Eed -/-
chip antibody: Ring1B
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 antibodies (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).
Antibody was received from Atsuta, T. et al. Production of monoclonal antibodies against mammalian Ring1B proteins. Hybridoma 20, 43-6 (2001)
Growth protocol All cells were cultured in KO DMEM supplemented with 10% FCS, 5% KO serum replacement (Invitrogen), Leukemia-Inhibitor factor, 50 µg/ml penicillin/streptomycin, 2 mM L-glutamine, 1x non-essential amino-acids and 50 µM 2-mercaptoethanol.
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 Genome Analyzer II
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
BAM index file for Eed WT = Eed_WT.bam
BAM index file for pooled Eed KO replicates = Eed_KO.bam
BAM index file for pooled Input replicates = Input.bam
GFF Peak calls using MACS = Eed_WT.gff3
GFF Peak calls using MACS = Eed_KO.gff3
Submission date Aug 19, 2010
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 GPL9250
Series (1)
GSE23716 H3K27me3 is not required for recruitment of Polycomb repressor complex 1 to target loci in mouse embryonic stem cells
SRA SRX028531
BioSample SAMN00115725

Supplementary data files not provided
SRA Run SelectorHelp
Processed data are available on Series record
Raw data are available in SRA

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap