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Status |
Public on Mar 21, 2016 |
Title |
Wapal shRNA#2-3 |
Sample type |
SRA |
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Source name |
ESC infected with Wapal shRNA #2
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Organism |
Mus musculus |
Characteristics |
cell type: murine ESC-129 derived infected with: Wapal shRNA #2 chip-antibody: anti-Ring1b (Active Motif 39664)
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Treatment protocol |
Cells were infected with two different shRNAs to Wapal, or an Empty Vector control. ESCs were infected and selected with puromcyin for 48hrs prior to chromatin generation
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Growth protocol |
Standard ESC conditions
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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 anti-Ring1b (Active Motif 39664) was added to chromatin extracts and incubated overnight. Protein A/G Dynal beads were added for 2 hours and beads were isolated with a magnet and then washed with 0.1% SDS Lysis Buffer, Low Salt Buffer ( 0.1% SDS, 1% Triton X-100, 2mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), LiCl Wash Buffer (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). NEB ChIp-seq library prep kit (cat# E7645)
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina NextSeq 500 |
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Description |
processed data file: W_RING1B_VS_BOTHINPUT_normalized.wig
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Data processing |
Samples were sequenced on Illumina HiSeq 2000 First 5 bases were trimmed for all the reads using Trimmomatic software and kept 49bp in length 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 Wiggle files were normalized by dividing each fragment pile up by total number of mapped reads in million Genome_build: MM9 Supplementary_files_format_and_content: wig files were generated using MACS14 1.4.2. Data values represent normalized pile up.
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Submission date |
Mar 02, 2016 |
Last update date |
May 15, 2019 |
Contact name |
Sridhar Rao |
E-mail(s) |
sridhar.rao@bcw.edu
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Phone |
4149373841
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Organization name |
BloodCenter of Wisconsin
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Department |
Blood Research Institute
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Street address |
8727 Watertown Plank Road
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City |
Milwaukee |
State/province |
WI |
ZIP/Postal code |
53226 |
Country |
USA |
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Platform ID |
GPL19057 |
Series (2) |
GSE63325 |
The cohesin associated factor Wapal is required for proper polycomb-mediated gene silencing |
GSE78826 |
The cohesin associated protein Wapal is required for proper polycomb-mediated gene silencing [Ring1b ChIP-seq] |
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Relations |
BioSample |
SAMN04528738 |
SRA |
SRX1609492 |
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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