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| Status |
Public on Jun 23, 2017 |
| Title |
S2_scRNA_Trp_10_R1 |
| Sample type |
SRA |
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| Source name |
S2 - Schneider cells
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| Organism |
Drosophila melanogaster |
| Characteristics |
cell line: S2 (Schneider cells)
sample type: short RNA sequencing
treatment: Triptolide inhibition
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| Treatment protocol |
Transcription inhibition was performed as previously described (Henriques et al. 2013). Triptolide and/or flavopiridol were added at a final concentration of 10uM(BM to the culture media for the indicated time (0, 2.5, 5, 10, 20, 40 minutes). Heat shock was performed in S2 cells as previously described (Wirbelauer et al. 2005). Temperature was quickly raised by mixing equivalent amount of cell containing medium at 25C(BC with pre-warmed medium (BC). The mix was incubated for 1 min at 37C(BC. After heat shock (HS), cells were let to recover for 5 min.Transcription inhibition (Trp, Fl) inhibition was performed 2.5 minutes prior heat-shock.
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| Growth protocol |
Schneider S2 cells were grown at 25 degrees in Schneider’s Drosophila medium (LifeTech:21720-001) supplemented with 10%FBS. OSC cells were grown at 25 degrees in Shields and Sang M3 Insect Medium (Sigma, S8398) supplemented with 1% insulin, 1% glutathione, 10% heat-inactivated FBS, and 10% Fly Extract.
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| Extracted molecule |
total RNA |
| Extraction protocol |
scRNA protocol was adapted fron Nechaev et al, Science 2010.For each condition 20 10^6 of Triptolide treated drosophila cells were spiked with 2 10^6 untreated mouse embryonic stem cells. scRNA protocol was adapted from (Nechaev et al., 2010). Cells were re-suspended in ice-cold lysis buffer (10mM Tris (pH=7.4), 10mM NaCl, 3mM Mgcl2, 0.1mM EDTA, 0.5% NP40), incubated 10min on ice, span down. Nuclei were washed with ice cold (10mM Tris (pH=7.4), 10mM NaCl, 3mM Mgcl2, 0.1mM EDTA) and nuclear pellets were dissolved in Trizol (Thermofisher). RNA was size selected (17-200bp) using a two-step column purification strategy (RNA clean and concentrator, Zymo-R1016). 10μg of purified RNA was successively treated by 5’ dephosphorylation - 20U at 37°C for 30min (Epicentre - RP8092H); 5’ terminator exonuclease - 1U in Buffer A at 30°C for 60min (Epicentre - TER51020); cap-clip decapping enzyme – 5U at 37°C for 90min. After each reaction, short RNA was column purified (RNA clean and concentrator, Zymo-R1016). The resulting RNA was used for library preparation using TruSeq small RNA library (Illumina). Libraries were purified on 6% TBE gels (150-300bp – Novex - EC6265BOX). Size distribution of the libraries were controlled on Bioanalyser High sensitivity (Agilent 5067-4626). Two biologically independent inhibition time courses were performed. The samples were run on an Illumina NextSeq generating 38bp paired-end reads.
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| Library strategy |
ncRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
nascent RNA
short RNA sequencing
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| Data processing |
After alignment against drosophila and mouse genomes (see above for details). Reads were counted in a window around TSSs [-100:200] using the qCount function of QuasR (Gaidatzis et al., 2014). To reduce potential contamination with full-length transcripts we used the second read from the pair representing the 3’ of the transcript for counting. Pairs having insert length>180bp were excluded. We used the mouse spike-ins to perform inter-sample normalization and enable quantification of the global effects expected to occur upon inhibition of transcription. Under the Assumption that read counts from the mouse should be constant across samples; reads counts from each fly sample were normalized by the sum of the reads detected at mouse promoters within the same sample. Abundance of scRNA at each time-point was calculated relative to the respective amount before inhibition. This relative level was calculated for each replicate separately to correct for batch variations.
Genome_build: Dm3
Supplementary_files_format_and_content: bigwig files
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| Submission date |
Jun 14, 2017 |
| Last update date |
Mar 23, 2020 |
| Contact name |
Dirk Schuebeler |
| Organization name |
Friedrich Miescher Institute for Biomedical Research
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| Street address |
Maulbeerstrasse 66
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| City |
Basel |
| ZIP/Postal code |
4058 |
| Country |
Switzerland |
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| Platform ID |
GPL19132 |
| Series (1) |
| GSE77369 |
Genome-wide single molecule footprinting reveals high RNA polymerase II turnover at paused promoters |
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| Relations |
| BioSample |
SAMN07236709 |
| SRA |
SRX2917419 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2668008_S2_Trp_10_R1.wig.gz |
588.7 Kb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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