|
| Status |
Public on Nov 05, 2013 |
| Title |
IMR90 BrdU-Seq |
| 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: BrdU (BD Biosciences, 347580)
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
|
| |
|
| 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 |
SRX212675 |
| BioSample |
SAMN01880850 |
