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Status |
Public on Mar 21, 2011 |
Title |
ES_RNApolII |
Sample type |
SRA |
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Source name |
Embryonic Stem Cells
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Organism |
Mus musculus |
Characteristics |
strain: C58BL/6 Bruce 4 (B4) cell type: ES cells chip antibody: RNA polymerase II C-terminal domain chip antibody vendor: Abcam chip antibody catalog#: ab5131
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Growth protocol |
Mouse C57BL/6 Bruce 4 (B4) ES cells were adapted to culture without helper fibroblasts and expanded using standard techniques. mESCs were cultured in DMEM (Invitrogen) with 15% (v/v) FCS (batch tested for ES cell culture), 100 ug/ml streptomycin, 100 IU/ml penicillin, beta-mercaptoethanol (Sigma), non-essential amino acids, Glutamax (Gibco) and recombinant mLIF 103 IU/ml (Millipore). To prepare chromatin for ChIP, mESCs were grown to ~80% confluence and fixed with buffered formaldehyde (1%) for 10 minutes at room temperature. G1ME cells were grown in alpha-MEM (Invitrogen) supplemented with 20% (v/v) FCS, 100 ug/ml streptomycin, 100 IU/ml penicillin and 70 ng/ml recombinant thrombopoietin (Stachura, 2006). For ChIP studies G1ME cells were grown at 5x105 cells/ml and were fixed with 1% formaldehyde for 20 minutes at room temperature.
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Extracted molecule |
genomic DNA |
Extraction protocol |
Chromatin was prepared from fixed mouse ES cells and G1ME cells and was sonicated with a Bandelin sonicator (30% amplitude, 15-25 30 second bursts with a 2 minute reprieve) to produce fragments from 200-1000 bp, with peak signal between 200-500 bp. Approximately 2 x107 cell equivalents were used for each immunoprecipitation. 1.7% of the sample was removed for use as an input control. ChIP was performed as described previously (Lee, 2006), using antibodies towards H3K27Me3 (07-449, Millipore) or phosphorylated RNA polymerase C-terminal domain (ab5131, Abcam).
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina Genome Analyzer II |
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Data processing |
All reads were mapped to version 9 of the mouse reference genome (mm9) using bowtie version 0.11.3 with the default settings and output to SAM format. Any reads that appeared on the same strand at the same location more than 5 times was discarded to remove PCR artifacts (i.e. at most five reads were allowed to start at any one location).
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Submission date |
Mar 15, 2011 |
Last update date |
Jun 11, 2013 |
Contact name |
Matthew Daniel Young |
E-mail(s) |
myoung@wehi.edu.au
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Organization name |
Walter & Eliza Hall Institute of Medical Research
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Department |
Bioinformatics
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Street address |
1G Royal Parade
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City |
Parkville |
State/province |
Victoria |
ZIP/Postal code |
3052 |
Country |
Australia |
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Platform ID |
GPL9250 |
Series (2) |
GSE27967 |
ChIP-seq analysis reveals distinct H3K27me3 profiles associated with gene regulation [ChIP-seq] |
GSE27970 |
ChIP-seq analysis reveals distinct H3K27me3 profiles associated with gene regulation |
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Relations |
BioSample |
SAMN02196884 |
Supplementary file |
Size |
Download |
File type/resource |
GSM691592_ES.RNApol.sorted.bam |
386.1 Mb |
(ftp)(http) |
BAM |
Processed data provided as supplementary file |
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