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| Status |
Public on Sep 22, 2021 |
| Title |
D3_V5_ChIPSeq |
| Sample type |
SRA |
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| Source name |
ES-D3
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| Organism |
Mus musculus |
| Characteristics |
cell line: ES-D3
cell type: embryonic stem cells
genotype: wild type
chip antibody: anti- V5 (Invitrogen R960-25)
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| Treatment protocol |
Cells were cross-linked for 5 minutes at room temperature with 1% formaldehyde-containing medium; cross-linking was stopped by PBS-glycine (0.125 M final). Cells were washed twice with ice-cold PBS, scraped, centrifuged for 10 min at 4000 rpm, 4 'C and pellets flash-frozen in liquid nitrogen and stored at - 80 'C until lysed.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cell pellets were thawed in ice and resuspended in cell lysis buffer (5 mM PIPES, pH 8.0, 85 mM KCl, and 0.5% NP-40, 1 ml/15 cm plate) and incubated for 10 min on ice. Lysates were then centrifuged for 10 min at 4000 rpm. Nuclear pellets were resuspended in 6 volumes of sonication buffer (50 mM Tris-HCl, pH 8.1, 10 mM EDTA, 0.1% SDS), incubated on ice for 10 min, and sonicated to obtain DNA fragments below 2000 bp in length (Covaris S220 sonicator, 20% Duty factor, 200 cycles/burst, 100 peak incident power, 32 cycles of 20" on and 40" off). Sonicated lysates were cleared by centrifugation and 800 μg of chromatin was diluted in RIPA buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate, 140 mM NaCl) to a final concentration of 0.8 μg/μl, precleared with Protein A sepharose (GE Healthcare) for 2 hours at 4°C and immunoprecipitated overnight with 8 μg of antibodies. 4% of the precleared chromatin was saved as input. Immunoprecipitated DNA was purified with the Qiagen QIAquick PCR Purification Kit, eluted in 60 μl of water and used for library preparation.
Libraries were prepared with the Illumina ChIP-seq DNA sample preparation kit according to manufacturer instructions (for input DNA we used only 150 ng of DNA)
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Base-calling performed with CASAVA by the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley
ChIP-seq reads quality control performed with FastQC (version 0.10.1)
ChIP-seq reads were aligned to the mm10 genome assembly using bowtie (version 0.12.8) with the following configurations: -n 2 -m 1
ChIP-Seq peaks were called using MACS2 (version 2.1.0.20140616) with the following configurations: -t <sample.bam> -c <input.bam> -f BAM -g mm --nomodel --extsize 250
bigWig coverage files were generated with deepTools (version 2.4.1) with the following configurations: bamCoverage -of bigwig --binSize 50 --normalizeTo1x 2150570000 --extendReads 250 --ignoreDuplicates
Genome_build: mm10
Supplementary_files_format_and_content: ChIP-seq: tdf files were generated using igvtools (igvtools count -w 50 -e 200) / txt files are Partek detected enriched regions RAD23B vs. IgG); RNA-seq: text files contain CuffDiff results
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| Submission date |
Sep 06, 2019 |
| Last update date |
Sep 24, 2021 |
| Contact name |
Robert Tjian |
| E-mail(s) |
[email protected]
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| Phone |
(510) 642-0884
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| Organization name |
UC Berkeley
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| Department |
MCB
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| Lab |
Tjian Lab
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| Street address |
University of California, Berkeley 450 Li Ka Shing #3370
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| City |
Berkeley |
| State/province |
CA |
| ZIP/Postal code |
94720 |
| Country |
USA |
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| Platform ID |
GPL21103 |
| Series (1) |
| GSE137036 |
Klf5 confers embryonic and extra-embryonic bi-potential cell fate in early blastomeres and embryonic stem cells |
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| Relations |
| BioSample |
SAMN12709216 |
| SRA |
SRX6813447 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4066081_D3_V5.bw |
122.2 Mb |
(ftp)(http) |
BW |
| GSM4066081_D3_V5_peaks.txt.gz |
598 b |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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