|
| Status |
Public on Nov 26, 2018 |
| Title |
WT rep 2 |
| Sample type |
SRA |
| |
|
| Source name |
DT40 cell line, S2 cell line
|
| Organisms |
Drosophila melanogaster; Gallus gallus |
| Characteristics |
cell line: DT40
cell type: chicken B-cell line
genotype/variation: wild type
antibody: monoclonal hybridoma purified S9.6 antibody
spiked with: Drosophila S2 cells
|
| Growth protocol |
DT40 cells were grown at 37 degrees Centrigrade and 5% CO2 atmosphere, in RPMI 1640 with Glutamax (Gibco) supplemented with 7% FBS (Gibco), 3% chicken serum (Sigma) and penicillin-streptomycin. Drosophila S2 cells were grown in Insect-XPRESS Protein-Free Insect Cell medium with L-glutamine (Lonza), supplemented with 1% penicillin-streptomycin. S2 cells were grown in suspension, at 27 ºC, ambient CO2 and 105 rpm agitation.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Upon mixing the DT40 cells with the S2 cells (3.75:1 ratio) , the nuclear fraction was extracted (then treated with rnaseH for the control), genomic DNA containing containing DNA:RNA hybrid purified and fragmented with restriction enzyme digest. Soluble contaminating RNA was digested with Rnase A treatments and DNA:RNA hybrids immunoprecipitated with S9.6 antibody. Following low, high, LiCl and TE buffer washes of Protein-G captured immunocomplexes, DNA was eluted, phenol:chloroform purified and fragmented to 300 bp average size with Covaris M220
Libraries were prepared with NEBNext Ultra II DNA Library Prep Kit (New England Biolabs, E7645) as per provided instruction for low input DNA samples, from approximately 30 ng of immunoprecipateted DNA. Size selection was ommited and total of 7 PCR amplification cycles.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
processed data file:
DT40_WT_broad_peaks.tsv
SLX-15836_S3
|
| Data processing |
library strategy: DNA-RNA immunoprecipitation (DRIP)-seq
Reads were quality trimmed and filtered for adaptor sequences using trim galore v0.4.4
Reads were aligned to Ggal 5.0 and Dmel r6.18 using bowtie2 v2.2.6
Uniquely mapping reads were separated into those aligning to the sample and the spike-in
Peaks were called using MACS2 v2.1.1.20160309 with options -g 1.87e9 –broad
Genome_build: Ggal 5.0 and Dmel r6.18
Supplementary_files_format_and_content: Tab-delimited files produced by MACS2 containing processing metadata and per peak information
|
| |
|
| Submission date |
Apr 05, 2018 |
| Last update date |
Nov 26, 2018 |
| Contact name |
Alastair Crisp |
| Organization name |
MRC Laboratory of Molecular Biology
|
| Street address |
Francis Crick Avenue
|
| City |
Cambridge |
| ZIP/Postal code |
CB2 0QH |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL24851 |
| Series (2) |
| GSE112746 |
Genome wide mapping of R-loops in the repriming deficient DT40 cells using DRIP-Seq |
| GSE112747 |
Repriming limits R-loop formation at DNA secondary structures during S phase |
|
| Relations |
| BioSample |
SAMN08871778 |
| SRA |
SRX3892925 |
