 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Apr 14, 2020 |
| Title |
WIZ Rep 1 |
| Sample type |
SRA |
| |
|
| Source name |
Mouse embryonic stem cells v6.5
|
| Organism |
Mus musculus |
| Characteristics |
genotype: Wild Type
cell line: mESC v6.5
antibody: anti-WIZ; Novus NBP1-80586
|
| 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)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
mESCs were crosslinked in 1% formaldehyde for 20 minutes then quenched with 125 mM glycine. Cells were lysed with Lysis Buffer 1 (50 mM Hepes-KOH pH7.5, 140 mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, and 0.25% Triton X-100) by incubating cells in the buffer for 10 minutes at 4C. Nuclei were next lysed with Lysis Buffer 2 (10mM Tris-HCl pH 8, 200mM NaCl, 1mM EDTA, and 0.5 mM EGTA) by incubating nuclei in the buffer for 10 minutes at room temp. Finally, nuclear extracts were resuspended in Sonication Buffer 1 (20mM Tris-HCl pH 8, 150mM NaCl, 2mM EDTA, 0.1% SDS, and 1% Triton X-100). Cells were sonicated using a Branson probe sonicator with the following settings: 18% amplitude, 30 sec on, 60 sec off, 17 cycles, 8.5 minutes total. WIZ antibody (4ug, NBP1-80586) was incubated with Protein G Dynabeads (150ul, Thermo Fisher, 10004D) for 6 hours at 4C. Unbound antibody was removed by washing beads three times with PBS before sonicated chromatin was added to antibody conjugated beads and incubated overnight at 4C. Beads were washed with sonication buffer, wash buffer 1 (20mM Tris-HCl pH 8, 500mM NaCl, 2mM EDTA, 0.1% SDS, and 1% Triton X-100), wash buffer 2 (10mM Tris-HCl pH 8, 250mM LiCl, 1mM EDTA, and 1% NP-40), and wash buffer 3 (10mM Tris pH 8, 1mM EDTA, and 50mM NaCl). Chromatin was eluted from beads by adding elution buffer (50mM Tris pH 8, 10mM EDTA, and 1% SDS) and incubating at 65C for 1 hour, spinning down the mixture, then moving the supernatant to a new tube. Supernatant was left at 65C overnight to reverse crosslinks. RNA was degraded by adding TE and RNAse A to the tubes at 37C for 2 hours followed by protein degradation with CaCl2 and Proteinase K for 30 minutes at 55C. DNA was precipitated using phenol chloroform followed by NaCl, glycogen, and ethanol addition. Resulting DNA pellet was resuspended in 10 mM Tris HCl pH 8. ChIP-seq library was prepared using the ThruPLEX-FD Prep kit (Takara, R400428).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
WIZ-ChIPseq.bw
|
| 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.
|
| |
|
| Submission date |
Sep 11, 2019 |
| Last update date |
Apr 14, 2020 |
| Contact name |
Jill Dowen |
| E-mail(s) |
jilldowen@unc.edu
|
| Organization name |
UNC Chapel Hill
|
| Street address |
250 Bell Tower Drive, CB 7100 3360 Genome Sciences Building
|
| City |
Chapel Hill |
| State/province |
NC |
| ZIP/Postal code |
27599 |
| Country |
USA |
| |
|
| 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 |
|
| Relations |
| BioSample |
SAMN12730508 |
| SRA |
SRX6831864 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
