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Sample GSM1050291

Query DataSets for GSM1050291

Status

Public on Feb 20, 2013

Title

Sox2 ESC ChIP-Seq

Sample type

SRA

Source name

Embryonic stem cells, Sox2 ChIP

Organism

Mus musculus

Characteristics

strain: C57BL/6:129 SvJae F1
cell type: embryonic stem cells (ESCs)
passage: 21
chip antibody: Sox2
antibody vendor, catalog#: R & D Systems (R&D), AF2018

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

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.
The processed data for this sample is included in GSE35496_Sox2_ESC_Wig_min0.1.WIG.gz, which is linked to the Series GSE35496 record as a supplementary file.

Submission date

Dec 07, 2012

Last update date

May 15, 2019

Contact name

Michael Lodato

E-mail(s)

lodato@wi.mit.edu

Organization name

Whitehead Institute

Street address

9 Cambridge Center