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Sample GSM1705254 Query DataSets for GSM1705254
Status Public on Nov 21, 2015
Title Naive_CTCF_R2
Sample type SRA
Source name Embryonic stem cells
Organism Homo sapiens
Characteristics cell type: Naive
chip antibody: CTCF
antibody manufacturer: Millipore
antibody catalog: 07-729
young_id: 20150318_3596
system: Embryo
Treatment protocol None
Growth protocol Naive hESCs were cultured as previously described (Theunissen et al., 2014). For the induction of naive hESCs, primed hESCs were cultured for 24 hr in the primed hESC medium described above, further supplemented with 10 μM ROCK inhibitor Y- 27632 (Stemgent, 04-0012). Colonies were then trypsinized to form a single cell suspension and cells were plated onto a MEF feeder layer in the primed hESC medium + ROCK inhibitor described above. 24 hr later, the medium was switched to 5i/L/A naive hESC medium. The 5i/L/A naive hESC medium (500 ml) used for induction and maintenance of naive hESCs was made up of 240 ml DMEM/F12, 240 ml Neurobasal (Invitrogen, 21103), 5 ml N2 supplement (Invitrogen, 17502048) and 10 ml B27 supplement (Invitrogen, 17504044), supplemented with 10 μg recombinant human LIF (purified in-lab from E. coli), 1 mM glutamine, 1% nonessential amino acids, 0.1 mM β- mercaptoethanol, penicillin-streptomycin, 50 μg/ml BSA (Sigma, A4737-25G), and the following small molecules and cytokines: 1 μM PD0325901 (Stemgent, 04-0006), 1 μM IM-12 (Enzo, BML-WN102-0005), 0.5 μM SB590885 (R&D systems, 2650/10), 1 μM WH-4-023 (A Chemtek) 10 μM Y-27632 (Stemgent, 04-0012), and 10 ng/ml Activin A (Peprotech, 120-14). Following an initial wave of widespread cell death, dome-shaped naive hESC colonies appeared within 10 days and could be expanded and maintained in 5i/L/A naive hESC medium.
Naive hESCs were maintained on mitomycin C-inactivated MEF feeder cells and passaged every 5-7 days. The naive hESCs were passaged by dissociating cells with accutase (GIBCO, A1110501), and then centrifuging cells at 1000 rpm for 5 minutes at room temperature in neutralization medium (DMEM supplemented with 10% FBS, 1 mM glutamine, 1% nonessential amino acids, penicillin-streptomycin, and 0.1 mM β- mercaptoethanol). To harvest cells for downstream experiments, primed and naive hESCs were trypsinized and subsequently pre-plated on gelatin-coated dishes to deplete MEF feeder cells. All cell culture experiments were performed under physiological oxygen conditions (5% O2, 3% CO2).
Extracted molecule genomic DNA
Extraction protocol Whole cell extracts were sonicated to solubilize the chromatin. The chromatin extracts containing DNA fragments with an average size of 500 bp were immunoprecipitated using different antibodies. Purified immunoprecipitated DNA were prepared for sequencing according to a modified version of the Solexa Genomic DNA protocol. Fragmented DNA was end repaired and subjected to 18 cycles of LM-PCR using oligos provided by Illumina. Amplified fragments between 150 and 300bp (representing shear fragments between 50 and 200nt in length and ~100bp of primer sequence) were isolated by agarose gel electrophoresis and purified.
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 2000
Data processing Aligned using /usr/local/bin/bowtie with configuration -p 4 --best -k 2 -m 2 --sam -l 40
genome build: hg19
Supplementary_files_format_and_content: WIG files(s) represent counts of aligned reads within 50 bp bins with each read being extended 200 in the direction of alignment. Counts are in reads-per-million and floored at 0.1
Submission date Jun 08, 2015
Last update date May 15, 2019
Contact name Richard A Young
Phone 617-258-5219
Organization name Whitehead Institute for Biomedical Research
Lab Young Lab
Street address 9 Cambridge Center
City Cambridge
State/province MA
ZIP/Postal code 02142
Country USA
Platform ID GPL11154
Series (2)
GSE69646 3D Chromosome Regulatory Landscape of Human Pluripotent Cells [ChIP-Seq]
GSE69647 3D Chromosome Regulatory Landscape of Human Pluripotent Cells
BioSample SAMN03764712
SRA SRX1053365

Supplementary file Size Download File type/resource
GSM1705254_20150318_3596.4310.rpm.WIG.gz 82.6 Mb (ftp)(http) WIG
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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