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Sample GSM970264 Query DataSets for GSM970264
Status Public on Oct 11, 2013
Title CTCF_ChIPSeq_Undifferentiated
Sample type SRA
Source name Undifferentiated mouse ES cells
Organism Mus musculus
Characteristics cell type: mouse ES cells
passage: 29
strain: E14 [129P2/OlaHsd]
chip antibody: CTCF (Millipore, 07-729, lot DAM1682158)
Growth protocol E14 mouse ES cells were grown in Dulbecco’s Modified Eagle’s Medium, High Glucose (DMEM, GIBCO® #11965-084), 15% Fetal Bovine Serum (FBS, HyClone #SH30071.03), 2 mM L-Glutamine (GIBCO® #25030-081), 0.1% 2-Mercaptoethanol (GIBCO® #21985-023) and 1000 u/ml ESGRO® supplement containing Leukemia Inhibitory Factor (Chemicon/Millipore #ESG1107), on mitomycin C treated mouse embryonic fibroblasts (MEF) plated on gelatinized cell culture plates. Prior to differentiation and to collection for RNA or chromatin preparation, ES cells were dissociated into single cell suspension using 0.25% Trypsin-EDTA (GIBCO® #25200056), and adsorbed twice, for 45 minutes each time, to remove MEF. Residual MEF contamination was estimated to be below 0.25% by visually inspecting using a hematocytometer. For ES cells differentiation, ES cells were plated at 10E4 cells/cm2 on gelatinized cell culture plates and grown in DMEM, 10% FBS, 2 mM glutamine, 0.1% 2-Mercaptoethanol and 0.1 µM all-trans-Retinoic acid (Sigma #R2625). After 4.5 days, cells were dissociated using 0.25 % Trypsin-EDTA and collected for chromatin or RNA preparation. In the cells collected for ChIP-seq and RNA-seq, cell death was estimated around 2% in undifferentiated cells and 8% in differentiated cells, using Trypan Blue staining.
Extracted molecule genomic DNA
Extraction protocol Chromatin immunoprecipitation was performed as previously described (Nelson & Denisenko, Nat Protoc. 2006;1(1):179-85). After cross-linking cells for 15 min with 1% formaldehyde and sonicating chromatin with a Diagenode Bioruptor, chromatin was immunoprecipitated using 10 µg of CTCF antibody (Millipore 07-729, lot DAM1682158) or 10 µg of IgG (Santa Cruz Biotechnology, Sc-2027) for 50E6 cells. In each case, for both undifferentiated and differentiated cells, ~15 ng of CTCF or IgG immunoprecipitated DNA were recovered after combining two technical replicates from the same biological sample. DNA fragments of ~150-300 bp range were isolated by agarose gel purification, ligated to primers, and then subject to Solexa sequencing using manufacturer's recommendations (Illumina, Inc.).
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina Genome Analyzer IIx
Description ctcf_peaks_undifferentiated.tsv
Data processing The image analysis and base calling of ChIP-seq data was performed using the Illumina Pipeline software.
Sequence alignment to the mm9 mouse genome was performed using Bowtie (Langmead et al., Genome Biol. 2009;10(3):R25).
Only uniquely aligned sequence tags, with up to two mismatches, were considered for further analysis. Tags aligned to blacklisted areas (the ENCODE consortium blacklist was downloaded from were also removed before the peak calling step.
The significant peak identification was performed using a two steps procedure, as previously described (Pal et al., Genome Res. 2011 Aug;21(8):1260-72). In the first step, statistically significant enriched genomic regions (of length 1 kb) were identified. A region is defined as statistically significant if the difference in number of reads between experiment (CTCF) and control (IgG) samples within the region is higher than a given cutoff read count, calculated using a p-value of 0.001. Each ChIP-Seq read distribution in the genome can be considered as a Poisson distribution and the difference of two Poisson distributions is given by the Skellam distribution. Skellam probability mass function is given by equation 1 (sup. file “Equations.pdf”). For each of the statistically significant enriched regions, ChIP-Seq reads overlapping profiles were created by extending the sequence reads from the 5' end to the 3' end of the reads up to 250 bp (the average length of the ChIP-DNA fragment sequenced from the Solexa GA with Illumina standard ChIP-seq protocol) for the experiment sample. Then, within each enriched region, the significant enriched peaks in experimental data are identified based on a threshold read count, obtained using a cutoff p-value ≤ 0.01.
Peak score was calculated within significant regions by moving average of extended CTCF ChIP-Seq reads in 500bp windows.
Genome_build: mm9
Supplementary_files_format_and_content: The "ctcf_peaks_*.tsv" files list CTCF peaks position in mouse mm9 genome assembly (columns 1 and 2) and, for each peak, CTCF peak score (column 3), the tag count for CTCF immunoprecipitation (column 4) and the tag count for the IgG control (column 5). The files "ctcf_peaks_undifferentiated.tsv" and "ctcf_peaks_differentiated.tsv" are for undifferentiated and differentiated cells, respectively. The "*_reads_*.tsv" files list reads position in the mouse mm9 genome assembly (columns 1-3), with their orientation (column 4). The files "ctcf_reads_undifferentiated.tsv" and "ctcf_reads_differentiated.tsv" list reads from CTCF ChIP-Seq in undifferentiated and differentiated cells, respectively. The files "igg_reads_undifferentiated.tsv" and "igg_reads_differentiated.tsv" list reads from the IgG ChIP-seq control in undifferentiated and differentiated cells, respectively. The "ctcf_undifferentiated.wig" and "ctcf_differentiated.wig" files show CTCF binding profiles after substraction of IgG background, in undifferentiated and differentiated cells, respectively.
Submission date Jul 19, 2012
Last update date May 15, 2019
Contact name Sebastien Vigneau
Phone +1-857-540-5439
Organization name Dana-Farber Cancer Institute
Department Cancer Biology
Lab Alexander Gimelbrant
Street address 450 Brookline Avenue
City Boston
State/province MA
ZIP/Postal code 02115
Country USA
Platform ID GPL11002
Series (2)
GSE39502 Differences in CTCF binding site sequence are associated with unique regulatory and functional trends during embryonic stem cell differentiation [ChIP-Seq]
GSE39523 Differences in CTCF binding site sequence are associated with unique regulatory and functional trends during embryonic stem cell differentiation
SRA SRX160828
BioSample SAMN01091702

Supplementary file Size Download File type/resource
GSM970264_ctcf_reads_undifferentiated.tsv.gz 121.2 Mb (ftp)(http) TSV
GSM970264_ctcf_undifferentiated.wig.gz 24.6 Mb (ftp)(http) WIG
SRA Run SelectorHelp
Processed data provided as supplementary file
Processed data are available on Series record
Raw data are available in SRA

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