|
| Status |
Public on Jan 22, 2019 |
| Title |
4SU-controlKD_NT_Rep1 |
| Sample type |
SRA |
| |
|
| Source name |
cell culture
|
| Organism |
Drosophila melanogaster |
| Characteristics |
pulldown: Biotin enrichment
cell type: Embryonic
cell line: S2R+ Cells
genotype/variation: control
treatment: Non DRB treated Ctrl Sample
|
| Treatment protocol |
dsRNA was transfected in S2R+ cells by serum starvation for 6 hours. The treatment was repeated three times and cells were harvested 7 days after the first treatment.
|
| Growth protocol |
Drosophila S2R+ cells were cultured in Schneider Cell’s Medium (GIBCO, Cat No-21720) supplemented with 10% FBS and 2% Penicillin/Streptomycin
|
| Extracted molecule |
total RNA |
| Extraction protocol |
5,6-dichlorobenzimidazole 1-_-d-ribofuranoside (DRB) from Sigma (D1916) was used at a final concentration of 300_M, dissolved in water, for 5 hours. 4-thiouridine (4sU) was purchased from Sigma (T4509) and used at a final concentration of 100 _M. Control and Mago KD was performed as described before. All the samples were labeled for 8 minutes with 4-thiouridine, and transcription was allowed to proceed after DRB removal for 0, 2, 8 and 16 minutes along with one non-DRB treated control.A total of 100 to 130 ug RNA was used for the biotinylation reaction. 4sU-labeled RNA was biotinylated with EZ-Link Biotin-HPDP (Pierce), dissolved in dimethylformamide (DMF) at a concentration of 1 mg/mL. Biotinylation was done in labeling buffer (10 mM Tris pH 7.4, 1 mM EDTA) and 0.2 mg/mL Biotin-HPDP for 2 h with rotation at room temperature. Two rounds of chloroform extractions removed unbound Biotin-HPDP. RNA was precipitated at 20,000 g for 20 min at 4¡C with a 1:10 volume of 5M NaCl and an equal volume of isopropanol. The pellet was washed with 75% ethanol and precipitated again at 20,000 g for 10 min at 4¡C. The pellet was left to dry, followed by resuspension in 100 _L RNase-free water. Biotinylated RNA was captured using Dynabeads MyOne Streptavidin T1 beads (Invitrogen). Biotinylated RNA was incubated with 50 _L Dynabeads with rotation for 15 min at 25¡C. Beads were magnetically fixed and washed with 1_ Dynabeads washing buffer. RNA-4sU was eluted with 100 _L of freshly prepared 100 mM dithiothreitol (DTT), and cleaned on RNeasy MinElute Spin columns (Qiagen).
Enriched nascent RNAs were converted to cDNA libraries with Drosophila Ovation Kit (Nugen) with integrated ribosomal depletion workflow
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Non DRB treated Ctrl Sample
|
| Data processing |
The data was sequenced on a HiSeq2500 in a rapid run with a readlength of 68 bp single read (HiSeq Rapid SBS/Cluster Kit v2) . Demultiplexing and fastq file conversion was done using blc2fastq v. 1.8.4.
Mapping was performed suing STAR (v. 2.5.1b) against ensembl release 84 of BDGP6.
UCSC compatible library normalised bigwig tracks were produced using samtools version 1.2, ucsc tools (bedGraphToBigWig from 04-11-2012) and bedtools (v. 2.25.0) and custom scripts
Genome_build: BDGP6
Supplementary_files_format_and_content: bigwig
|
| |
|
| Submission date |
Dec 14, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Jean-Yves Roignant |
| Organization name |
Institute of molecular Biology
|
| Lab |
Roignant
|
| Street address |
Ackermannweg 4
|
| City |
Mainz |
| ZIP/Postal code |
55128 |
| Country |
Germany |
| |
|
| Platform ID |
GPL17275 |
| Series (2) |
| GSE92379 |
DRB-4sU seq in control and Mago depleted S2R+ cells [Promoter-proximal pausing mediated by the exon junction complex regulates splicing] |
| GSE92389 |
Promoter-proximal pausing mediated by the exon junction complex regulates splicing |
|
| Relations |
| BioSample |
SAMN06141217 |
| SRA |
SRX2422459 |
