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Sample GSM1552409 Query DataSets for GSM1552409
Status Public on May 06, 2015
Title rsEpiSCs-input
Sample type SRA
Source name rsEpiSCs
Organism Mus musculus
Characteristics cell type: E5.75 derived rsEpiSCs
passages: 20-25
strain: OCT4-deltaPE-GFP x C57BL/6
chip antibody: none
Growth protocol Mouse EpiSCs were grown on MEFs in N2B27 media containing 12ng/ml FGF2 and 20ng/ml Activin-A supplementated with 20% KSR; rsEpiSCs were grown on MEFs in N2B27 media containing 20ng/ml and IWR1 2.5uM; Human H1 ESCs were grown on Matrigel-coated plates in mTeSR1 media, Human H1 rsESCs were grown on Matrigel-coated plates in customized mTeSR1 media containing 20ng/ml FGF2 and 2.5uM IWR1.
Extracted molecule genomic DNA
Extraction protocol Cells were fixed with 1% formaldehyde at 37C for 10min, quenched with glycine at 37C for 5 min. Fixed cells were sonicated using Epishear (Active Motif) to achieve 200-700 size chromatin fragments. Solubilized chromatin was immunoprecipiated with antibody against H3K4me3 (Abcam 8580) and H3K27me3 (Millipore 07-449). Antibody-chromatin complexes were pulled down using Dynabeads protein A (Invitrogen), washed and then eluted. After cross-linking reversal, RNase and proteinase K treatment, immunoprecipiated DNA was purified using AMPure beads (Beckman Coulter).
ChIP DNA were end-repaired and 5' phosphorylated using T4 DNA Polymerase, Klenow and T4 Polynucleotide Kinase (Enzymatics). A single Adenine was added to 3' ends by Klenow (3-->5' exo-), and double-stranded Bioo Illumina Adapters (Bioo Scientific) were ligated to the ends of the ChIP fragments. Adapter-ligated ChIP DNA fragments were subjected to 15 cycles of PCR amplification using Q5 polymerase (NEB). AMPure beads were used to purify DNA after each step (Beckman Coulter). Pooled libraries were sequenced on the NextSeq 500 for single-end 75bp using high-output flowcell or on the HiSeq 2000 for single-end 36bp according to manufacturer’s instructions.
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina NextSeq 500
Description Input for ChIP-Seq in rsEpiSCs
Data processing Basecalls performed using CASAVA version 1.8.2.
ChIP-Seq reads were aligned to the mouse (mm9) and human (hg19) genomes using bowtie2 (v2.1.0), keeping only reads that mapped to a unique genomic location (MAPQ > 10).
HOMER (v4.6) was used to process alignment files to generate normalized bedGraphs and bigWigs for the UCSC Genome Browser and quantify H3K4me3 and H3K27me3 levels at promoters.
Genome_build: MGSCv37 (mm9)
Supplementary_files_format_and_content: bigWig format. Initial reads were extended to approximately 150 bp and normalized coverage bedGraphs were generated using HOMER. The bedGraphToBigWig utility from the UCSC Genome Browser was then used to generate the bigWig files.
Submission date Nov 21, 2014
Last update date May 15, 2019
Contact name Christopher Benner
Organization name University of California, San Diego (UCSD)
Department Medicine
Street address 9500 Gilman Dr. MC 0640
City La Jolla
State/province California
ZIP/Postal code 92093-0640
Country USA
Platform ID GPL19057
Series (2)
GSE60604 An alternative pluripotent state confers interspecies chimaeric competency [ChIP-seq]
GSE60605 An alternative pluripotent state confers interspecies chimaeric competency
BioSample SAMN03216766
SRA SRX835948
Named Annotation GSM1552409_LP-EpiSCs-input.ucsc.bigWig

Supplementary file Size Download File type/resource
GSM1552409_LP-EpiSCs-input.ucsc.bigWig 208.3 Mb (ftp)(http) BIGWIG
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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