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| Status |
Public on Jan 19, 2022 |
| Title |
Day10 hMEC #2 |
| Sample type |
SRA |
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| Source name |
human mammary epithelial cells
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| Organism |
Homo sapiens |
| Characteristics |
day: 10
antibody: anti-CTCF (Millipore)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIPs were performed essentially as described [82] with minor modifications. Unless stated otherwise, all chemicals were purchased from Sigma. Briefly, normal HMECs and stressed HMECs were crosslinked for 15 minutes by addition of formaldehyde (Invitrogen) to a 1% final concentration. Crosslinking was stopped upon addition of glycine to a final concentration of 125 mM for 5 minutes. After washing with 1X PBS, cells were scraped and collected in RIPA (150 mM NaCl, 1% NP-40, 0.5% Sodium Deoxycholate, 0.1% SDS, 50 mM Tris-HCl pH 8.0, 5 mM EDTA) containing protease inhibitors. Lysates were subsequently sonicated and 500 mg of lysate was pre-cleared for 1 hour using 40 ml of a 50% slurry of 1:1 protein A- and protein G-Sepharose beads (GE Healthcare). For immunoprecipitation, anti-CTCF (Millipore) was added to precleared lysates along with 40 ml of a 50% slurry of 1:1 protein A/G beads and incubated at 4°C overnight. Immuno-complexes were washed with: RIPA; IP wash buffer (100 mM Tris-HCl pH 8.5, 500 mM LiCl, 1% NP-40, 1% Sodium Deoxycholate); followed by 1X TE buffer before eluting in 200 ml of buffer containing 70 mM Tris-HCl pH 8.0, 1 mM EDTA and 1.5% SDS after incubation at 65°C for 10 or 30 minutes. Crosslinks were reversed from immuno-complexes by addition of 200 mM NaCl and incubation at 65°C for 6 hours or overnight. DNA was purified by incubation with proteinase K and phenol-chloroform extraction. Input samples were treated identically and IP DNA was quantified by real time PCR (RT-PCR).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
updatedCTCDiff.txt
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| Data processing |
Sequenced reads were quality-tested using FASTQC [85]
The genome index was constructed using the gene annotation supplied with the hg19 Illumina iGenomes collection and sjdbOverhang value of 100.
Reads were aligned to the hg19human genome using the STAR [86] version 2.5.1b. Mapping was carried out using default parameters (up to 10 mismatches per read, and up to 9 multi-mapping locations per read).
ChIP-Seq analysis was carried out using HOMER findPeaks and mergePeaks subroutines using default parameters (four-fold enrichment over local tag count, Poisson p-value < 0.001, and style factor).
Peaks common to replicate 1 and replicate 2 at day 1 or day 10 were kept, and merged into a common peak file. Raw read counts were then assigned to peaks using HOMER annotatePeaks using all replicates from both days.
Differential CTCF peaks were found using raw counts of merged peaks from both day 1 and day 10 HMECs with DESeq2 using parameters of FDR < 0.05 and log2fold > 1.
Normalized read densities were visualized using the UCSC genome browser [90].
Genome_build: hg19
Supplementary_files_format_and_content: Text file containing differential peak quantification and testing with HOMER
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| Submission date |
Nov 04, 2019 |
| Last update date |
Mar 28, 2022 |
| Contact name |
April Elizabeth Williams |
| E-mail(s) |
apriljack06@gmail.com, awilliams@salk.edu
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| Phone |
7345461645
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| Organization name |
Salk Institute for Biological Studies
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| Department |
IGC
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| Street address |
10010 N Torrey Pines Rd
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| City |
San Diego |
| State/province |
California |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE139882 |
Dynamic regulation of stress-responsive non-genomic CTCF complexes [ChIP-seq] |
| GSE139886 |
Dynamic regulation of stress-responsive non-genomic CTCF complexes |
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| Relations |
| BioSample |
SAMN13191072 |
| SRA |
SRX7098805 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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