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Status |
Public on May 13, 2020 |
Title |
24_p5424_K27ac |
Sample type |
SRA |
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Source name |
p5424
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Organism |
Mus musculus |
Characteristics |
tissue source: cell line - p5424 thymocyte genotype: CTCF Motif WT protocol: Cut and Run-seq rnp: No RNP chip antibody: H3K27ac ab Ab4729 1ug
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Extracted molecule |
genomic DNA |
Extraction protocol |
Cells were washed 1X with cut and run wash buffer (20 mM HEPES, pH7.5, 150 mM NaCl, 0.5 mM spermidine), bound to activated ConA beads (Bangs Laboratories BP531), permeabilized in digi buffer (wash buffer + 0.002% digitonin), incubated with antibodies (1 g/CR in digi buffer), washed in digi buffer, incubated with pA-MN (gift from Heinkoff Laboratory, currently EpiCypher 15-1016), and washed in digi buffer. Following the final wash, cells were washed with ice-cold low salt wash buffer (20 mM HEPES, pH 7.5, 0.5 mM spermidine, 0.002% digitonin) and digested using MN-ase digestion buffer (3.5 mM HEPES pH 7.5, 10 mM CaCl2, 0.002% digitonin) for 25 minutes on ice. Solubilized chromatin was released using an isosmotic stop buffer (170 mM NaCl, 20 mM EGTA, 0.05% Digitonin, 20 µg/ml glycogen, 25 µg/ml RNase A, 2 pg/ml S. cerevisiae fragmented nucleosomal DNA) and was collected using a PCR clean up kit column (EZbioresearch M1001). All antibody incubations and digitonin washing steps were performed for 5 minutes at room temperature and included protease, phosphatase and deacetylase inhibitors (Roche). For library preparation, DNA products were incubated with an end repair master mix (1 X T4 ligation buffer, dNTP, ATP, T4 PNK, T4 DNA Pol and TAQ DNA polymerase) and incubated as follows: 12’C 15 minutes (end polishing), 37’C 15 minutes (5’ end phosphorylation) and 58’C 1.5 hour (dA tailing). Polished libraries were purified using AmpureXP beads and ligated to either NEBNext Dual Index (NEB) or Illuminia TruSeq adaptors. Libraries were size selected and enriched by PCR. Finished libraries were sequenced using Illumina HiSeq2500 (Genome Technology Access Center, Washington University).
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Library strategy |
OTHER |
Library source |
genomic |
Library selection |
other |
Instrument model |
Illumina HiSeq 2500 |
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Data processing |
Unique reads were aligned to the reference build (GTCm387/mm9) using TopHat and Bowtie2. duplicates were removed using Picard markDuplicates RPKM values were obtained using Deeptools. peaks were called using MACS2 findpeaks The reads were aligned to the mm9 build using Bowtie2 to map clean reads to reference gene Genome_build: mm9 Supplementary_files_format_and_content: RPKM values were determined using STAR aligner.
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Submission date |
May 12, 2020 |
Last update date |
May 14, 2020 |
Contact name |
Patrick Leonard Collins |
E-mail(s) |
patrick.collins@osumc.edu
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Phone |
6156683538
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Organization name |
Ohio State University
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Department |
Microbial Infection and Immunity
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Street address |
770 BRT, 460 W 12th Ave
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City |
Columbus |
State/province |
OH |
ZIP/Postal code |
43210 |
Country |
USA |
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Platform ID |
GPL17021 |
Series (1) |
GSE150384 |
DNA Double-Strand Breaks Induce H2Ax Phosphorylation Domains in a Contact-Dependent Manner |
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Relations |
BioSample |
SAMN14897260 |
SRA |
SRX8330550 |
Supplementary file |
Size |
Download |
File type/resource |
GSM4548047_24_p5424_K27ac.bw |
73.0 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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