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Sample GSM4548047 Query DataSets for GSM4548047
Status Public on May 13, 2020
Title 24_p5424_K27ac
Sample type SRA
 
Source name p5424
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
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).
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina HiSeq 2500
 
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.
 
Submission date May 12, 2020
Last update date May 14, 2020
Contact name Patrick Leonard Collins
E-mail(s) patrick.collins@osumc.edu
Phone 6156683538
Organization name Ohio State University
Department Microbial Infection and Immunity
Street address 770 BRT, 460 W 12th Ave
City Columbus
State/province OH
ZIP/Postal code 43210
Country USA
 
Platform ID GPL17021
Series (1)
GSE150384 DNA Double-Strand Breaks Induce H2Ax Phosphorylation Domains in a Contact-Dependent Manner
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 SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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