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Sample GSM1545804 Query DataSets for GSM1545804
Status Public on Mar 21, 2016
Title Wapal_rep1
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 SAMN03196582
SRA SRX760389

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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