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Sample GSM2078262 Query DataSets for GSM2078262
Status Public on Mar 21, 2016
Title Wapal shRNA#2-2
Sample type SRA
 
Source name ESC infected with Wapal shRNA #2
Organism Mus musculus
Characteristics cell type: murine ESC-129 derived
infected with: Wapal shRNA #2
chip-antibody: anti-Ring1b (Active Motif 39664)
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
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 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)
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina NextSeq 500
 
Description processed data file: W_RING1B_VS_BOTHINPUT_normalized.wig
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.
 
Submission date Mar 02, 2016
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 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]
Relations
BioSample SAMN04528737
SRA SRX1609491

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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