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| Status |
Public on Apr 14, 2020 |
| Title |
CTCF 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-CTCF; Active Motif 61311
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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 for 2 minutes before quenching with 125 mM glycine. Crosslinked cells were lysed using Lysis Buffer 1 and Lysis Buffer 2 before resuspension in Sonication Buffer 1. Human chromatin (from HEK293T cells) was spiked in (to final 5%) prior to sonication for the indicated experiments. Sonication of nuclei was performed on a Covaris E220 with the following settings: Duty Factor 8, PIP/W 210, and 200 cycles per burst for 12 minutes. Chromatin fragments of 200-1,000 base pair size were generated. Antibody was incubated with 100uL Protein G Dynabeads for 6 hours. Unbound antibody was removed via washing, as detailed above, before incubation of antibody bound beads with chromatin overnight. Beads were then washed with Sonication Buffer 1 and Wash Buffers 1, 2, and 3. Chromatin was eluted and crosslinks were reversed overnight via incubation at 65C and addition of 5ul Proteinase K. Zymo ChIP DNA Clean and Concentrate kit (D5205) was used to purify DNA following Proteinase digestion. Sequencing libraries were prepared using a Hyper Prep kit (Kapa Biosciences KK8502).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
CTCF-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 |
GPL21103 |
| 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 |
SAMN12730506 |
| SRA |
SRX6831869 |
| 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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