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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 (http://liulab.dfci.harvard.edu/MACS/) 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

E-mail(s)

stephen.taylor@imm.ox.ac.uk

Phone

+44 1865 222640

Organization name

CBRG