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| Status |
Public on Apr 14, 2020 |
| Title |
RAD21 WIZdel Rep 2 |
| Sample type |
SRA |
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| Source name |
Mouse embryonic stem cells v6.5
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| Organism |
Mus musculus |
| Characteristics |
genotype: WIZ deletion
cell line: mESC v6.5
antibody: anti-RAD21; Bethyl A300-080A
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| Growth protocol |
V6.5 murine embryonic stem cells (mESCs) (male) were grown on irradiated murine embryonic fibroblasts under standard conditions, as previously described (Dowen et al, 2014)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
mESCs were crosslinked in 1% formaldehyde (ThermoFisher, 28906) for 5 minutes in PBS and quenched by adding glycine. Cells were lysed using Lysis Buffers 1 and 2, and recovered nuclei resuspended in 1ml Covaris Sonication Buffer (50mM Tris pH 7.5, 10mM EDTA, 0.1% SDS). Human HEK293T nuclei prepared in the same manner were spiked-in to a final concentration of 5%, and nuclei were sheared for 12 minutes using a Covaris E220 water bath sonicator with device settings of duty factor 5, PIP/W of 140, and 200 cycles per burst. Insoluble material was cleared by spinning sonicated lysates for 10 minutes at 21,000rcf. 10µg anti-RAD21 antibody (Bethyl, A300-080A) was incubated for 8 hours with 30µl of Dynabeads in PBS, after which excess antibodies were washed from the beads. 107 cell equivalents of sheared chromatin were incubated overnight with the antibody-bound beads in 1 ml Sonication Buffer 1, washed with Wash Buffers 1, 2, and 3, and crosslinks were reversed overnight. DNA was purified using a ChIP DNA Clean and Concentrator Kit (Zymo, D5201) according to manufacturer’s instructions. Sequencing libraries were prepared using a Hyper Prep kit (Kapa Biosciences, KK8502) according to manufacturer’s instructions.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
For RAD21 ChIPs, only the first read of the pair was used for analysis. Thus, the two samples with labels "R1" were concatenated, and the first 50 bp were used for subsequent analysis.
Rad21-WIZdel-ChIPseq.bw
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| Data processing |
Alignment: reads were aligned to a hybrid mm10+hg38 genome with bowtie v 1.2.2 with parameters -v 2 -p 24 -S -m 1 --best --strata
PCR Duplicate Removal: duplicates removed with samtools v 1.9 fixmate -m followed by markdup -r -s
Peak Calling: peaks were called using MACS (v 2016-02-15) with parameters -f BED -g mm -q 0.01
Spike-In Normalization Factor: reads mapping to each genome (mouse and human) were counted. Normalization factor = 1/(number human reads / 1,000,000)
Coverage File Generation: bigwig files were generated using bedtools (v 2.26) genomecov with parameters -bga -g mm10.chrom.sizes -scale (Normalization Factor) followed by ucsctools (v 320) bedGraphToBigWig
Genome_build: Custom built bowtie index of Mus musculus build mm10 plus Homo sapiens build hg38
Supplementary_files_format_and_content: For each ChIPped factor, a BigWig coverage track of merged replicates is provided for viewing in UCSC genome browser.
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| Submission date |
Sep 11, 2019 |
| Last update date |
Apr 14, 2020 |
| Contact name |
Jill Dowen |
| E-mail(s) |
jilldowen@unc.edu
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| Organization name |
UNC Chapel Hill
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| Street address |
250 Bell Tower Drive, CB 7100 3360 Genome Sciences Building
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| City |
Chapel Hill |
| State/province |
NC |
| ZIP/Postal code |
27599 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE137272 |
A WIZ/Cohesin/CTCF complex anchors DNA loops to define gene expression and cell identity [ChIP-Seq] |
| GSE137285 |
A WIZ/Cohesin/CTCF complex anchors DNA loops to define gene expression and cell identity |
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| Relations |
| BioSample |
SAMN12730504 |
| SRA |
SRX6831871 |
| 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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