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| Status |
Public on Oct 11, 2013 |
| Title |
Input_ChIPSeq_Undifferentiated_Rep2 |
| Sample type |
SRA |
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| Source name |
Undifferentiated mouse ES cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: mouse ES cells
passage: 29
strain: E14 [129P2/OlaHsd]
chip antibody: none
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| Growth protocol |
Protocol for the undifferentiated ES cells biological replicate: 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 collection for 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.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Protocol for the undifferentiated ES cells biological replicate: Chromatin immunoprecipitation was performed as previously described (Nelson & Denisenko, Nat Protoc. 2006;1(1):179-85). After cross-linking cells for 10 min with 1% formaldehyde and sonicating chromatin with a Diagenode Bioruptor, chromatin was immunoprecipitated using 25 µl (~25 µg) of CTCF antibody (Millipore 07-729, lot 2054523) or 25 µg of IgG (Santa Cruz Biotechnology, Sc-2027) for 20E6 cells. 10 ng of CTCF immunoprecipitated DNA, IgG immunoprecipitated DNA or input were used for library preparation according to NEBNext ChIP-Seq library protocol (NEB #E6200S/L) using AMPure XP Beads (Agencourt #A63880) and with 14 cycles PCR enrichment using index primers for multiplexing. Indexes were as follows: CTCF, acagtg; IgG, cgatgt; input, tgacca. DNA fragment size was verified using a Bioanalyzer (Agilent #5067-4626) and the libraries were sequenced as 50 bp single-reads with the Illumina hiSeq sequencing system.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| 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 http://genome.ucsc.edu/cgibin/hgFileUi?db=hg19&g=wgEncodeMapability) 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 read 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 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: Description for the undifferentiated ES cells biological replicate: The file “ctcf_peaks_undifferentiated_rep2.tsv” file lists CTCF peaks position in mouse mm9 genome assembly (columns 1 to 3) and enrichment score (column 4). The “ctcf_reads_undifferentiated_rep2.bed”, “igg_reads_undifferentiated_rep2.bed” and “input_reads_undifferentiated_rep2.bed” files list reads position in the mouse mm9 genome assembly (columns 1-3), with their orientation (column 6), for CTCF, IgG and input samples. The “ctcf_undifferentiated_rep2.wig” and “igg_undifferentiated_rep2.wig” files show CTCF binding and IgG background profiles.
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| Submission date |
Aug 19, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Sebastien Vigneau |
| E-mail(s) |
sebastien_vigneau@dfci.harvard.edu
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| Phone |
+1-857-540-5439
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| Organization name |
Dana-Farber Cancer Institute
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| Department |
Cancer Biology
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| Lab |
Alexander Gimelbrant
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| Street address |
450 Brookline Avenue
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| 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 |
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| Relations |
| BioSample |
SAMN02319251 |
| SRA |
SRX337186 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1212179_input_reads_undifferentiated_rep2.bed.gz |
157.6 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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