 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Mar 21, 2016 |
| Title |
Wapal_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
Embryonic stem cells
|
| Organism |
Mus musculus |
| Characteristics |
cell type: ESC
cell type modification: engineered to express a biotinylatable version of Wapal
chromatin pulldown: Streptavidin Dynal Beads (Life Techniologies, Cat #65601)
|
| Treatment protocol |
ESCs were engineered to express a biotinylatable version of Wapal
|
| Growth protocol |
Standard ESC conditions
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Briefly, cells were crosslinked for 5 min at room temperature with 1% fresh formaldehyde. Reaction was quenched with glycine (125 mM final concentration) for 5 min at room temperature. Cells were lysed in 0.1% SDS lysis buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH8.0,150 mM NaCl). Chromatin was sonicated to a mean size of 300-500bp. Lysed cells were cleared by centrifugation and added to to 50 μL of Streptavidin Dynal Beads (Life Techniologies, Cat #65601). ψBeads were isolated with a magnet and then washed with SDS Buffer ( 2% SDS) x2, High Salt ( 0.1% Deoxycholate, 1% Triton X-100, 1mM EDTA, 50 mM Hepes, 7.5, NaCl 500 mM), LiCl wash (250 mM LiCl, 0.5% NP-40, 0.5% Deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0), and TE x2. DNA was eluted and decrosslinked overnight at 65 oC in SDS Elution Buffer (1% SDS, 10mM EDTA, 50 mM Tris-HCl pH 8.0). The next day RNase A and proteinase K were added, and the DNA was precipitated in the presence of glycogen. DNA for downstream applications were quantitated by flourometry (Qubit, Invitrogen), and equal DNA was used for each ChIP-qPCR and normalized to sheared, non-IP’d genomic DNA (Input).
The BioScientific NEXTFlex ChIp-seq kit was used.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Samples were sequenced on Illumina HiSeq 2000
Alignment to the mouse genome build mm9 was performed using Bowtie with parameters -n 2 -q -m 1 -l 49 -e 70 -k 1 --best
Peak calling was done using MACS14 1.4.2 with pvalue 1e-5
Genome_build: mm9
Supplementary_files_format_and_content: bed and Wiggle files were generated using MACS14
|
| |
|
| Submission date |
Nov 16, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Sridhar Rao |
| E-mail(s) |
sridhar.rao@bcw.edu
|
| Phone |
4149373841
|
| Organization name |
BloodCenter of Wisconsin
|
| Department |
Blood Research Institute
|
| Street address |
8727 Watertown Plank Road
|
| City |
Milwaukee |
| State/province |
WI |
| ZIP/Postal code |
53226 |
| Country |
USA |
| |
|
| Platform ID |
GPL13112 |
| Series (2) |
| GSE63324 |
The cohesin offloading factor Wapal is required for proper polycomb-mediated gene silencing [ChIP-Seq] |
| GSE63325 |
The cohesin associated factor Wapal is required for proper polycomb-mediated gene silencing |
|
| Relations |
| BioSample |
SAMN03196581 |
| SRA |
SRX760390 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
