|
|
GEO help: Mouse over screen elements for information. |
|
Status |
Public on Nov 30, 2011 |
Title |
Eed WT Input DNA replicate 3 |
Sample type |
SRA |
|
|
Source name |
Embryonic stem cells
|
Organism |
Mus musculus |
Characteristics |
strain: C57BL/6 cell type: embryonic stem cells genotype/variation: wild type chip antibody: no IP
|
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
|
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 |
|
Relations |
SRA |
SRX028535 |
BioSample |
SAMN00115729 |
Supplementary data files not provided |
SRA Run Selector |
Processed data are available on Series record |
Raw data are available in SRA |
|
|
|
|
|