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| Status |
Public on Feb 01, 2024 |
| Title |
JM8.N4_C59_Smc3_ChIPSeq |
| Sample type |
SRA |
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| Source name |
embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
cell line: JM8.N4
genotype: FLAG-Halo-CTCF/Rad21-SNAPf-V5 Knock-in
chip antibody: Smc3 (Bethyl A300-060A)
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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).
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| Growth protocol |
JM8.N4 mouse embryonic stem cells were grown on plates pre-coated with a 0.1% autoclaved gelatin solution (Sigma-Aldrich, G9391) under feeder free condition in knock-out DMEM with 15% FBS and LIF (full recipe: 500 mL DMEM (ThermoFisher#10829018), 6 mL MEM NEAA (ThermoFisher #11140050), 6 mL GlutaMax (ThermoFisher#35050061), 5 mL Penicillin-streptomycin (ThermoFisher #15140122), 4.6 μL 2-mercapoethanol (Sigma-Aldrich M3148), 90 mL fetal bovine serum (HyClone FBS SH30910.03 lot #AXJ47554)). mES cells were fed by replacing half the medium with fresh medium daily and passaged every two days by trypsinization.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were washed twice with ice-cold PBS, scraped, centrifuged for 10 min at 4000 rpm, 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. During the incubation, the lysates were repeatedly pipetted up and down every 5 minutes. 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, 50 cycles of 30" on and 30" off). Sonicated lysates were cleared by centrifugation and 400-1600 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 8-16 μg of normal rabbit IgGs, anti-Rad21 or anti-CTCF antibodies. About 15% 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 analyzed by qPCR together with 2% of the input chromatin prior to ChIP-Seq library preparation.
ChIP-seq libraries were prepared using the Illumina TruSeq™ DNA sample preparation kit according to manufacturer instructions with few modifications. We used 100 ng of ChIP input DNA (as measured by Fragment analyzer™) and 50 μl of immunoprecipitated DNA as a starting material; library samples were enriched through 12 cycles of PCR amplification. We assessed library quality and fragment size by qPCR and Fragment analyzer™. We sequenced 7 multiplexed libraries per lane on the Illumina HiSeq4000 sequencing platform (single end-reads, 50 bp long) at the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley, supported by NIH S10 OD018174 Instrumentation Grant.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
Homozygote cell line with endogenously tagged CTCF and Rad21 loci
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| Data processing |
Base-calling performed with CASAVA by the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley
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: .bed files containing ChIP-Seq peak genomic coordinates (mm10), length, summit, pileup, pvalue, qvalue and fold enrichment over input as determined by MACS2
Supplementary_files_format_and_content: bigWig files containing the number of reads per 50 bp bins normalized to 1x sequencing depth (Reads Per Genomic Content)
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| Submission date |
Sep 19, 2017 |
| Last update date |
Feb 01, 2024 |
| Contact name |
Robert Tjian |
| E-mail(s) |
jmlim@berkeley.edu
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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) |
| GSE104000 |
Genome-wide binding profile of Halo-tagged cohesin |
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| Relations |
| BioSample |
SAMN07666134 |
| SRA |
SRX3197481 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2787574_C59_Smc3_coverage.bw |
149.3 Mb |
(ftp)(http) |
BW |
| GSM2787574_C59_Smc3_peaks_25734.bed.gz |
613.4 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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