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| Status |
Public on Mar 21, 2016 |
| Title |
Input1-3 |
| Sample type |
SRA |
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| Source name |
Sheared Chromatin (biological replicate 1)
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| Organism |
Mus musculus |
| Characteristics |
cell type: murine ESC-129 derived
infected with: None
chip-antibody: None
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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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| 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 |
SAMN04528746 |
| SRA |
SRX1609500 |
| 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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