|
| Status |
Public on Jan 04, 2013 |
| Title |
PSC_mitotic_S2_ChIPSeq replicate 1 |
| Sample type |
SRA |
| |
|
| Source name |
S2 cell line
|
| Organism |
Drosophila melanogaster |
| Characteristics |
cell line: S2
chip antibody: PSC custom made rabbit polyclonal antibody that was affinity purified (described in Francis et al., 2009. PMID: 19303136)
facs sorting antibody: H3S10p
cell cycle profile: mitotic
|
| Treatment protocol |
for mitotic samples S2 or S2-PH cells were treated with 350ng/ml (880nM) colchicine for 15hrs.
|
| Growth protocol |
Drosophila S2 cells were grown in ESF921 medium (Expression Systems) at 27°C
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were crosslinked with 1% formaldehyde for 10 min at room temperature. Crosslinking was quenched with glycine. Cells were stained with antibodies to either anti-H3S10p (colchicine-treated cells) or anti-H3 (asynchronous cultures) that were either FITC-conjugated or treated with a FITC-conjugated secondary antibody and FACS sorted to collect FITC positive cells. Nuclear lysates were sonicated to generate 300-600 bp DNA fragments. Chromatin was incubated overnight with antibody against PSC at 4°C (S2 cells) or without antibody (S2-PH cells). Chromatin was precipitated with streptavidin-coated beads for 2 hours at 4°C. After washing and eluting from beads the crosslinking was reversed and DNA was isolated. Eluted DNA and input DNA was sheared with a Bioruptor (Diagenode) to give ~180bp fragments. Libraries were generated by the BioMicroCenter (MIT) using the SPRI-works system.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Alignment: Sequenced reads were aligned to the D. melanogaster genome (dm3) using Bowtie 1.1.2 (Langmead, B. et al., 2009) with the paramters -n 2 -m 1 --best --strata through Galaxy (Blankenberg, D. et al., 2010; Goecks, J. et al., 2010; Giardine, B. et al., 2005).
Peaks: peak detection was performed using the Model-Based Analysis of ChIP-Seq (MACS, version 1.0.1) through Galaxy using --tsize 36, --gsize 120000000 --bw 200 --nomodel --shiftsize 100. Peaks were filtered to retain peaks with a false discovery rate of 5% or below for all samples except for mitotic PH. Peaks on chromosome U and Uextra were removed.
Genome_build: dm3
Supplementary_files_format_and_content: Wig files were generated using MACS (version 1.0.1) and represent shifted tag counts for every 10bp. Wig files were normalzed by reads per million (RPM). Bed files contain a score that is given by -10*LOG10(pvalue).
|
| |
|
| Submission date |
May 23, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Nicole Follmer |
| E-mail(s) |
[email protected]
|
| Organization name |
Harvard University
|
| Street address |
16 Divinity Avenue
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02138 |
| Country |
USA |
| |
|
| Platform ID |
GPL13304 |
| Series (1) |
| GSE38166 |
Polycomb Group proteins are retained at specific sites on chromatin in mitosis |
|
| Relations |
| SRA |
SRX149189 |
| BioSample |
SAMN00997864 |
