|
| Status |
Public on Nov 05, 2013 |
| Title |
H1 Control BrdU-Seq |
| Sample type |
SRA |
| |
|
| Source name |
H1 embryonic stem cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Embryonic stem cells
culture medium: mTESR1 (STEMCELL Technologies)
passage: Between 35 to 45
antibody: BrdU (BD Biosciences, 347580)
cell line: H1
sirna treatment: Non-Targeting siRNA
|
| Treatment protocol |
siRNA treatment: EP300 and CREBBP siRNA (Thermo Scientific) was mixed with DharmaFECT 1 (Thermo Scientific) in Opti-MEM (Life Technologies). After incubation for 20 minutes at room temperature, the siRNA-DharmaFECT 1 complex was added to 6-well plates that were pre-coated with Matrigel (BD Biosciences). 300,000 of hESCs in single cell suspension were added to each well and mixed with transfection reagents. HA 1077 (EMD Millipore) was added to culture media to final concentration 10 µM. Same amount of non-targeting siRNA was used as control. Cells were collected at 72 hr post-transfection.
|
| Growth protocol |
H1 embryonic stem cells were cultured in mTESR1 medium. Cells were passaged every 5-6 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 |
SRX212679 |
| BioSample |
SAMN01880854 |
