|
| Status |
Public on Jan 09, 2016 |
| Title |
SIN3 220HA_HA_rep1_chipseq |
| Sample type |
SRA |
| |
|
| Source name |
S2 cells
|
| Organism |
Drosophila melanogaster |
| Characteristics |
cell type: Stably transfected HA-tagged SIN3 220 cells
cell line: S2
chip antibody: Anti-HA-Agarose antibody (Sigma)
|
| Growth protocol |
Drosophila S2 cells stably transfected with HA-tag constructs of SIN3 187 or SIN3 220 were cultured in Drosophila Schneider's media + L-glutamine supplemented with 10% heat inactivated fetal bovine serum and 0.1 mg/ml of penicillin/streptomycin and 0.1 mg/ml of geneticin. Cells were mainatined at 27oC
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin was prepared from cells which were treated with formaldehyde to chross-link protein and DNA. Fragmentation of chromatin to desired size was carried out by MNase treatment and sonication. Chromatin prepared was subjected to immunoprecipitation.
Libraries were prepared according to a previously published protocol (Ford et al.,2014). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation, 5 cycles of PCR was carried out using Illumina universal primers to convert all adapter DNA fragments to double stranded DNA. Following the pre_PCR step fragments of approximately 270 - 420 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. Purified DNA was PCR amplified with Illumina primers for 12 more cycle cycles. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the HiSeq 2000 following the manufacturer's protocols.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Basecalls performed using CASAVA version 1.8.2
ChIP-seq reads were aligned to the dm3 genome assembly using Bowtie2 with default parameters
Data were filtered using the following specification: 1. Reads mapping to unique region of the Drosophila genome, 2. phred score of reads > 20
peaks were called using MACS 2.1.0 and Irreproducibilty rate analysis was performed to call the final peaks. ChIP peak threshold (IDR 0.1), Enrichment Fold (≥3).
Genome_build: dm3
Supplementary_files_format_and_content: Bedgraph files were generated using MACS 2.1.0, Peak files were generated by MACS2.1.0 in bed format. Finally Peaks were called using IDR of 0.1 and FE ≥3
|
| |
|
| Submission date |
Aug 18, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Lori Ann Pile |
| E-mail(s) |
[email protected]
|
| Organization name |
Wayne State University
|
| Department |
Biological Sciences
|
| Street address |
5047 Gullen Mall
|
| City |
Detroit |
| State/province |
Michigan |
| ZIP/Postal code |
48202 |
| Country |
USA |
| |
|
| Platform ID |
GPL13304 |
| Series (2) |
| GSE72171 |
Mapping binding sites of SIN3 isoforms across the Drosophila genome. |
| GSE72173 |
Genome-wide studies reveal novel biological pathways regulated by SIN3 isoforms |
|
| Relations |
| BioSample |
SAMN03998767 |
| SRA |
SRX1158169 |
