|
| Status |
Public on Dec 11, 2012 |
| Title |
H1_input DNA |
| Sample type |
SRA |
| |
|
| Source name |
H1 human embryonic stem cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: H1
passages: 35-42
antibody: none
|
| Growth protocol |
H1 hESCs were plated on Matrigel (BD Biosciences)-coated plates, and maintained in mTeSR (StemCell). Before purification, cells were trypsinized to single cells and TRA-1-60 expressing cells were isolated by using MACS cell separation columns (Miltenyi Biotec). Isolated cells were tested by flow cytometry, and samples with >99% purity were used. IMR90 human primary lung embryo fibroblasts (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) plus 10% FBS (Hyclone), 100 U/ml penicillin (Gibco), and 100 µg/ml streptomycin (Gibco) at 37°C in 5% CO2. Growing cells with 50~70% confluence were used for further assay.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Standard chromatin immunoprecipiation protocol
~20 ng of ChIP and Input double strand DNA were end-repaired, added ‘A’ base to the 3-prime end, and ligated to adaptors by using Illumina ChIP-seq DNA Sample Prep. Kit Box 1. DNA fragments with 150 – 300 bp were selected and purified by agarose gel extraction, and amplified by PCR using Phusion polymerase (Illumina ChIP-seq DNA Sample Prep. Kit Box 1) according to manufacturer’ instrctions. Amplified DNA were purified by gel extraction and quantified by Qubit dsDNA BR assay (Invitrogen). DNA sequencing was performed by Illumina GAIIx sequencer with read length of 75 base pairs as per manufacturer’s protocol. Raw reads data were generated by the software SCS2.6. Further data analysis is available via online supporting information.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
Chromatin IP input DNA
|
| Data processing |
Unique sequences (duplicated sequences were removed and mapped only once to the genome) were aligned to human genome reference HG19 by using Bowtie-0.12.7 (command: bowtie -r -t -o 6 -a -m 1 --best --strata -v 2). Each chromosome was divided into windows of 100 bp. Number of reads in each window was calculated. To normalize total reads of ChIP enriched and Input DNA, the ratio of total reads from ChIP and Input DNA was calculated to generate a normalization factor which was applied to each value of the Input sample. To effectively capture local biases in the genome, we calculated the ratio of ChIP versus Input value in each window, and Poisson distribution was used to calculate a p-value for each window [2]. Significant peaks were defined as enrichment of ChIPed DNA over input DNA within a 100-base pair (bp) window at a Poisson p-value ≤ 0.001. Windows with significant p-values but with no neighboring significant peak or with no input reads were filtered out. To visualize large enrichment blocks, moving average was performed with moving window size of 10 kb and moving steps of 100 bp.
Genome_build: hg19
Supplementary_files_format_and_content: BED files containing genomic intervals of significant peaks
|
| |
|
| Submission date |
Jan 31, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Jing-Yu Li |
| E-mail(s) |
jingyuli@mednet.ucla.edu
|
| Organization name |
University of California, Los Angeles
|
| Street address |
615 Charles E. Young Dr. South BSRB 357
|
| City |
Los Angeles |
| ZIP/Postal code |
90095 |
| Country |
USA |
| |
|
| Platform ID |
GPL10999 |
| Series (2) |
| GSE26967 |
Dynamic Distribution of Linker Histone H1.5 in Cellular Differentiation |
| GSE26979 |
Human Linker Histone H1.5 |
|
| Relations |
| SRA |
SRX040573 |
| BioSample |
SAMN00205418 |
