|
| Status |
Public on Dec 21, 2017 |
| Title |
human_BJ_fibroblast_ChIP_Input_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
BJ foreskin fibroblasts
|
| Organism |
Homo sapiens |
| Characteristics |
antibody: none
sonication of purified dna prior to library prep: none
|
| Treatment protocol |
Cells at passage 12 were grown to ~80% confluence and crosslinked with 1% formaldehyde.
|
| Growth protocol |
Human BJ foreskin fibroblasts were obtained from Stemgent (08-0027) at passage 6 and cultured in Eagle’s Minimum Essential Medium (Sigma M2279) supplemented with 10% fetal bovine serum (FBS, Hyclone SH30071) and 2mM L-glutamine (Gibco) at 37°C and 5% CO2.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Crosslinked chromatin was sonicated using a Diagenode Bioruptor and sedimented on a 6-40% sucrose gradient for 3 hours at 41K rpm (see Methods of text for full details). Fraction #2 (near top of the gradient) containing highly sonicated fragments was harvested as the Euchromatin fraction, while Fractions #10-17 containing larger chromatin fragments was pooled as the Euchromatin fraction. Alternatively, chromatin was immunoprecipitated using Dynabead Protein G beads saturated with anti-H3K9me3 antibodies (abcam 8898, lot GR164997-3). H3K9me3-directed IPs were also performed using chromatin from the Euchromatin and Sonication-Resistant Heterochromatin fractions. DNA was purified by reversing crosslinks overnight, treating with RNase A and Proteinase K, and performing phenol-chloroform extraction.
50 ng of purified DNA was used to prepare libraries using the NEBNext Ultra DNA Library Prep Kit for Illumina (E7370S). For samples containing larger DNA fragments (gradient input, Sonication-Resistant Heterochromatin fraction, and IP from the Sonication-Resistant Heterochromatin fraction), the purified DNA was sheared to 100-300bp with a Covaris sonicator prior to library preparation.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Input chromatin used for H3K9me3 ChIP-seq studies
|
| Data processing |
Samples were demultiplexed (bcl2fastq using BaseSpace) to produce FASTQ files.
Sequenced reads were aligned to the hg19 assembly of the human genome using bowtie2 v2.1.0 (parameters: --very-sensitive). Bowtie output files were converted to .bam files using samtools v1.1, and then to .bed files using bedtools v2.20.1 (bamtobed).
For each sample, reads mapping to the same genomic position (duplicate reads) were collapsed to a single entry (unique reads).
75bp sequencing reads were extended to 200bp, to match the average size of DNA prior to library preparation.
To generate input-normalized genome coverage tracks, BED files were converted to BedGraph files using genomeCoverageBed (bedtools v2.20.1) and normalized to the number of millions of reads sequenced, to correct for lane or sample biases. For each sample’s normalized BedGraph, the corresponding input sample was subtracted on a basepair-by-basepair basis. The resulting subtracted BedGraph was converted to a bigWig file using bedGraphToBigWig (v4).
Genome_build: hg19
Supplementary_files_format_and_content: bigWig
|
| |
|
| Submission date |
Sep 16, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Gregory Donahue |
| Organization name |
The University of Pennsylvania
|
| Department |
Cell & Developmental Biology
|
| Lab |
Zaret Lab
|
| Street address |
3400 Civic Center Blvd, Bldg 421
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL18573 |
| Series (2) |
| GSE87039 |
Genomic and proteomic resolution of heterochromatin and its restriction of alternate fate genes (ChIP-seq) |
| GSE87041 |
Genomic and proteomic resolution of heterochromatin and its restriction of alternate fate genes |
|
| Relations |
| BioSample |
SAMN05785383 |
| SRA |
SRX2173005 |
