|
Status |
Public on Nov 05, 2013 |
Title |
IMR90 BrdU-Seq Input |
Sample type |
SRA |
|
|
Source name |
IMR90 fibroblasts
|
Organism |
Homo sapiens |
Characteristics |
cell type: Primary lung fibroblasts culture medium: DMEM supplemented with 10% FBS and 1X antibiotics passage: Between 7 to 12 antibody: None cell line: IMR90 sirna treatment: none
|
Growth protocol |
IMR90 cells were cultured in DMEM (Cellgro) that was supplemented with 10% FBS (Hyclone) and 1X antibiotic (Gibco). Cells were passaged every 3 days.
|
Extracted molecule |
genomic DNA |
Extraction protocol |
BrdU-Seq: 4 µg of BrdU labelled DNA was heated in 0.1 M NaOH at 95°C for 5 min to remove RNA primers on nascent strand DNA. HCl was then added to neutralize the pH. TE buffer (10 mM Tris-HCl pH 7.4, 1 mM EDTA) was added to bring the total volume 500 µl. DNA was fragmented by sonication and denatured by heating at 95°C for 5 min. 0.1 volume of Adjusting buffer (110 mM sodium phosphate buffer, pH7.0, 1.54 M NaCl, 0.55% Triton X-100) was then added to DNA. DNA was subsequently incubated with 2 µg of BrdU antibody (BD Biosciences, 347580) and Dynabeads Protein G (Life Technologies). After immunoprecipitation, DNA was purified using phenol/chloroform extraction and ethanol precipitation. Double-stranded DNA was generated from both input and immunoprecipitated DNA by brief random priming with the Bioprime DNA Labeling System (Life Technologies) at 37 °C for 20 min. Sequencing libraries were prepared with Chip-Seq DNA Sample Prep Kit (Illumina) according to the manufacturer's instructions.
|
|
|
Library strategy |
OTHER |
Library source |
genomic |
Library selection |
other |
Instrument model |
Illumina HiSeq 2000 |
|
|
Data processing |
Library strategy: BrdU-Seq
Reads were aligned to the human hg19 genome assembly using bowtie-0.12.7 with parameters -r --offrate 6 -m 1 --best --strata -t -v 2
peaks were called based on the p value for Poisson distribution of enriched immunoprecipitated DNA relative to input. Significant BrdU peaks were defined as those windows with a p value<10e-4 and with two neighboring windows at the same significance. Significant H3K18ac peaks were defined as those windows with a p value<10e-3 and with two neighboring windows at the same significance.
Genome_build: hg19
Supplementary_files_format_and_content: bed and wig files
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|
|
Submission date |
Dec 26, 2012 |
Last update date |
May 15, 2019 |
Contact name |
Siavash K Kurdistani |
E-mail(s) |
Skurdistani@mednet.ucla.edu
|
Organization name |
UCLA
|
Department |
Biological Chemistry
|
Lab |
Kurdistani
|
Street address |
615 Charles E Young Dr South
|
City |
Los Angeles |
State/province |
CA |
ZIP/Postal code |
90095 |
Country |
USA |
|
|
Platform ID |
GPL11154 |
Series (1) |
GSE43152 |
A unique epigenetic signature is associated with active DNA replication loci in human embryonic stem cells |
|
Relations |
SRA |
SRX212676 |
BioSample |
SAMN01880851 |