|
| Status |
Public on Feb 06, 2013 |
| Title |
Dm S2-DRSC ASH1N ChIP-seq |
| Sample type |
SRA |
| |
|
| Source name |
S2-DRSC cells
|
| Organism |
Drosophila melanogaster |
| Characteristics |
cell line: S2-DRSC cells
chip antibody: ASH1N
chip antibody source: Frank Sauer, Riverside
|
| Growth protocol |
S2-DRSC cells (DGRC lot 181A1) were grown at 25°C in Schneider's Drosophila medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (HyClone Cat No.: SH30070.03, Lot No. ASK30724).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin fixation and immunoprecipitation were performed essentially as described in DOI 10.1006/meth.1996.0407. In short, 1x10^9 cells were fixed in 200 ml medium with 1% formaldehyde for 10 min at room temperature. Cross-linked cells were sonicated to produce chromatin fragments of an average size of 200-400 bp. Soluble chromatin was separated from insoluble material by centrifugation. The supernatant containing chromatin of 2.5 x 10^7 cells was taken for immunoprecipitation. ASH1 bound chromatin was enriched using ASH1-N and ASH1-C polyclonal rabbit antibodies (kind gift from Frank Sauer, Riverside). Polyclonal rabbit antibodies for FSH were a gift from Igor Dawid, Laboratory of Molecular Genetics, PGD, NICHD. The antibody ID173 was raised against protein 4.1 (recognizes FSH-L only) and ID166 was raised against the N-terminal, common part of FSH-S and FSH-L, protein 1.2. The cDNAs used for the expression of both antigenes have been described in Digan ME et al. 1986. Libraries were prepared from ~10 ng of precipitated DNA using ChIP-seq DNA Sample Prep. Kit according to manufacturer’s instructions (Illumina, Cat# IP-102-1001). After adapter ligation library fragments of ~250 bp were excised from an agarose gel. The size selected DNA was PCR-amplified with Illumina primers for 18 (ChIP-seq) cycles, purified and loaded on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer IIx following the manufacturer's protocols.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Data processing |
Quality filtered reads were aligned to the Drosophila reference genome (BDGP Release 5) using Bowtie 0.12.7 and the following parameters: -n 2 -m 20 -k 1 --best (Langmead et al. 2009). Merging and indexing of alignments was done using SAMtools 0.1.9 (Li et al. 2009). Read coverage profiles and regions showing significantly enriched read coverage (peaks) were calculated using MACS 1.4.0 with the following parameters: band width=300, model fold=10 to 30, p-value cut-off=1x10^5 (Zhang et al. 2008). Alignments of sequenced input chromatin were used a control files during peak calling. Coverage profiles were calculated from shifted and extended reads according to the fitted MACS model.
BDGP Release 5
|
| |
|
| Submission date |
Mar 09, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Tobias Kockmann |
| E-mail(s) |
tobias.kockmann@bsse.ethz.ch
|
| Organization name |
ETHZ
|
| Department |
D-BSSE
|
| Lab |
Paro
|
| Street address |
Mattenstr. 26
|
| City |
Basel |
| ZIP/Postal code |
4058 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL11203 |
| Series (2) |
| GSE36404 |
Generation of chromatin maps for ASH1 and FSH applying ChIP-seq |
| GSE36450 |
The BET protein FSH functionally interacts with ASH1 to orchestrate global gene activity in Drosophila |
|
| Relations |
| SRA |
SRX128300 |
| BioSample |
SAMN00810066 |
