|
| Status |
Public on Oct 31, 2013 |
| Title |
Adelman_Dmel_S2_Mock-tr+FP_3prRNA |
| Sample type |
SRA |
| |
|
| Source name |
Drosophila S2 Cells short capped RNA
|
| Organism |
Drosophila melanogaster |
| Characteristics |
cell line: S2
|
| Treatment protocol |
RNAi was performed as described previously (Gilchrist et al. Genes Dev 2008). Cells were harvested 72 hours after addition of dsRNA. To block P-TEFb-mediated pause release, cells were treated with 500 nM Flavopiridol (Sigma) or DMSO vehicle for 10 minutes.
|
| Growth protocol |
Drosophila S2 cells from the DGRC were grown in M3 media supplemented with bactopeptone, yeast extract and 10% FBS. For all experiments, cells were harvested at a consistent cell density of 4-6x106 cells/ml.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from nuclei, whole cells, or fractionated cells as indicated using Trizol reagent (Invitrogen).
Liibraries were prepared for 3’ scRNA-sequencing as described in (Nechaev et al., Science 2010).
|
| |
|
| Library strategy |
ncRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
short capped RNA isolated from Drosophila S2 nuclei
|
| Data processing |
26 nt scRNA reads were aligned to the Drosophila dm3 genome with Bowtie, requiring unique alignment and allowing 2 mismatches (-m1 -v2).
The genomic location and strand of mapped reads were compiled using custom scripts and visually examined using the UCSC genome browser in .bedgraph format. RNA sequences came from discrete locations and were not binned for further analyses, allowing the hits to be examined at nucleotide resolution.
Normalization: to account for differential sequencing depth, all samples were normalized to 15 million reads. To enable accurate cross-sample comparisons of short capped RNA data sets, the following procedures were implemented. Seventeen synthetic short-capped RNA spikes (length 48-54 nt) were designed to align to non-TSS regions of the Drosophila genome that were systematically devoid of scRNA reads. RNAs were generated by in vitro reverse-transcription and capping. Each spike was added individually to each sample at the Trizol extraction stage at a constant ratio of either 1 or 10 spikes per genome (as determined by cellular or nuclear DNA yield). Counts for each spike scRNA were determined by summing the number of reads aligned in 100-nt strand-specific bins surrounding the genomic sequence complementary to the spike. Cross-sample comparisons were performed by calculating the ratio (aligned read count spiken sample 1)/(aligned read count spiken sample 2) for each of the 17 spikes. The spike normalization factor was calculated as the median of these ratios. For all scRNA samples except flavopiridol-treated samples, the spike normalization factor was ~1 and no spike-based normalization was performed. For three flavopiridol treatment experiments, the spike normalization factor ranged from ~0.6 to ~0.7, reflecting an increase in scRNA species in response to flavopiridol and a corresponding decrease in spike recovery. Flavopiridol-treated samples were normalized by dividing read counts by the respective normalization factors derived from median spike abundance.
Genome_build: dm3
Supplementary_files_format_and_content: Bedgraph files with unbinned normalized counts representing 3' ends of uniquely aligned scRNAs.
|
| |
|
| Submission date |
Jul 22, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Karen Adelman |
| E-mail(s) |
karen_adelman@hms.harvard.edu
|
| Organization name |
Harvard Medical School
|
| Department |
Biological Chemistry and Molecular Pharmacology
|
| Street address |
45 Shattuck St. LHRRB-201a
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL11203 |
| Series (1) |
| GSE49078 |
Stable pausing by RNA polymerase II provides an opportunity to target and integrate regulatory signals |
|
| Relations |
| Reanalyzed by |
GSM3284367 |
| BioSample |
SAMN02261491 |
| SRA |
SRX326850 |
