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Sample GSM1050312 Query DataSets for GSM1050312
Status Public on Feb 20, 2013
Title H3K27Ac ChIP-Seq Day2 rtta, rep1
Sample type SRA
Source name Day2 differentiating rtta ESCs, H3K27Ac ChIP
Organism Mus musculus
Characteristics strain: C57BL/6:129 SvJae F1
cell type: Day2 differentiating rtta ESCs
passage: 20-25
chip antibody: H3K27Ac
antibody vendor, catalog#: Abcam, ab4729
Growth protocol ESCs were grown under standard conditions in DMEM supplemented with 10% fetal bovine serum, LIF, glutamine, pen-strep, and beta-mercaptoethanol. NPCs were derived from these ESCs using an established protocol (PMID:8892235), then grown in N3 medium (see manuscript methods section). Day 2 differentiated cells were obtained from TetO-Brn2 and rtta ESCs plated in ESM with 2 ug/ml doxycycline, then 24 hours later the medium was switched to N2B27 plus doxycycline (see manuscript methods), and 48 hours after that cells were harvested for ChIP.
Extracted molecule genomic DNA
Extraction protocol For ESCs and Day 2 differentiating cells: Approximately 5x10^7 formaldehyde-crosslinked cells were lysed and as above on an IP-Star (Diagenode). Chromatin was sonicated on the Bioruptor (Diagenode) to an average size of 0.2-1 kb. ChIP was performed on chromatin from approximately 5 million cells with 3 μg of antibody (above) using the IP-Star Automated System (Diagenode), and 2.5% of chromatin was used for each whole cell extract (WCE). Following reversal of crosslinks, sample and WCE DNA was purified. ChIP DNA was dissolved in water and placed on the SPRI-TE (Beckman Coulter) for Illumina sample preparation. NPCs: ChIP-enriched and input DNA were purified and genomic libraries were prepared using the ChIP-Seq Sample Prep Kit (Illumina 1003473) according to the manufacturer's protocol (Illumina 11257047) for selecting library fragments between 200 and 350 bp.
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 2000
Data processing The processed data for this sample is included in GSE35496_H3K27Ac_rtta_Day2_Wig_min0.1.WIG.gz
Images acquired from the Illumina/Solexa sequencer were processed using the bundled Solexa image extraction pipeline.
36 bp or 40 bp sequences were aligned using Bowtie software (PMID:19261174) using murine genome NCBI Build 36 (UCSC mm8) as the reference genome with default settings for mismatch tolerance, non-unique mapping events, etc.
Sequences were extended +200 bp for transcription factors and allocated in 25 bp bins (1.05x10^8 bins total). Statistically significant enriched bins were identified using a Poissonian background model, generally with a p-value threshold of 10-8 to minimize false positives. We used an empirical background model (whole cell extracts (WCE)) that require genomic bins to be enriched at least 5-fold above background to correct for non-random enrichment observed previously.
Genome_build: MGSCv36 (mm8)
Supplementary_files_format_and_content: Wig files. One track for each dataset reflects ChIP-Seq density across the genome in 25bp bins with a minimum cutoff of 0.1 reads per million. Another track depicts enriched regions for each dataset at a p-value threshold of 10-8.
Submission date Dec 07, 2012
Last update date May 15, 2019
Contact name Michael Lodato
Organization name Whitehead Institute
Street address 9 Cambridge Center
City Cambridge
State/province MA
ZIP/Postal code 02142
Country USA
Platform ID GPL13112
Series (1)
GSE35496 ChIP-Seq of Sox2 and Brn2 in ESCs, NPCs, and differentiating ESCs
SRA SRX209337
BioSample SAMN01828236

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

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