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| Status |
Public on Nov 30, 2011 |
| Title |
EedWT-CBX7 |
| Sample type |
SRA |
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| Source name |
Embryonic stem cells WT
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| Organism |
Mus musculus |
| Characteristics |
cell type: embryonic stem cells
strain: C57BL/6
genotype/variation: wild type
chip antibody: CBX7
chip antibody vendor: Abcam
chip antibody catalog #: ab21873
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| 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).
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| 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.
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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 |
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
genome build: mm9
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| Submission date |
Oct 21, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Stephen Taylor |
| E-mail(s) |
[email protected]
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| Phone |
+44 1865 222640
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| Organization name |
CBRG
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| Street address |
Headington
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| City |
Oxford |
| ZIP/Postal code |
OX3 9DS |
| Country |
United Kingdom |
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| 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 |
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| Relations |
| SRA |
SRX101807 |
| BioSample |
SAMN00740098 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM820726_Eed_WT_CBX7.bam |
6.6 Gb |
(ftp)(http) |
BAM |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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