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| Status |
Public on May 01, 2013 |
| Title |
nLac.fastq.gz |
| Sample type |
SRA |
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| Source name |
S2_mock control
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| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: 24hr embryo
cell type: Schneider line 2 (S2) cells
treated with: dsRNA directed against E. coli LacI (Control)
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| Treatment protocol |
RNAi was performed as described, for 1 hour in serum-free media before addition of Chelex-100 treated complete media. Cells were incubated with dsRNA for 3.5 days. S2 cells were treated with 40 ug/ml of dsRNA against TAF1 or mock treated for 1 hour in the absence of serum.
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| Growth protocol |
Drosophila melanogaster Schneider line 2 (S2) cells were maintained at 25C in Schneider‘s Medium containing 10% FBS, 100 units/mL penicillin, and 0.1 mg/ml streptomycin.
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| Extracted molecule |
total RNA |
| Extraction protocol |
6x10^7 cells were pelleted (1500xg) and washed once with cold 1xPBS. Nascent RNA was harvested using the NUN RNA isolation protocol as described in Khodor YL, et al. (2011) with the following addition: after lysis and dounce homogenization in buffer AT (15 mM HEPES-KOH at pH 7.6, 10 mM KCl, 5 mM MgOAc, 3 mM CaCl2, 300 mM sucrose, 0.1% Triton X-100, 1 mM DTT, 1× SigmaFAST), nuclei were pelleted at 1000xg for 5 minutes and resuspended in 1 mL of fresh buffer AT prior to layering on Buffer B (15 mM HEPES-KOH at pH 7.6, 10 mM KCl, 5 mM MgOAc, 3 mM CaCl2, 1 M sucrose, 1 mM DTT, 1× SigmaFAST). RNA was purified from pellets in 1 mL of TriReagent.
Ribosomal RNA was depleted from 6 µg of nascent RNA as follows: 950 pmoles of a mixture of biotinylated oligos targeting ribosomal RNA (Table S3) were bound to 0.6 ml of Magnesphere Beads (Promega). Beads were washed 4 times with 0.5x SSC containing 10 mM EDTA. Oligo-bound beads were combined with nascent RNA in 2xSSC/10 mM EDTA and heated at 68C for 10 minutes followed by incubation at 25C for 15 minutesand held on ice for 2 hours with periodic mixing. Ribosomal-depleted RNA was collected. Beads were washed once with 0.5xSSC/10 mM EDTA. Ribosomal-depleted RNA from the supernatant and wash were combined and precipitated with alcohol.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
nascent transcript
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| Data processing |
fastx_clipper from fastx_toolkit-0.0.13.2 to remove the 3' adapter sequence with paramaters "-M 9 -n -l 20"
take the reverse complement sequences after 3' adapter is removed
Genome_build: Drosophila melanogaster, BDGP R5/dm3 (http://www.ncbi.nlm.nih.gov/assembly/GCA_000001215.2/)
Supplementary_files_format_and_content: tab delimited text format; 1st column: sequence (after trimming adapters and reverse-complement), 2nd column: frequency
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| Submission date |
Apr 08, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Michael Marr |
| E-mail(s) |
[email protected]
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| Organization name |
Brandeis University
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| Department |
Biology
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| Street address |
415 South St
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| City |
Waltham |
| State/province |
Massachusetts |
| ZIP/Postal code |
02454 |
| Country |
USA |
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| Platform ID |
GPL13304 |
| Series (1) |
| GSE45873 |
Holo-TFIID controls the magnitude of a transcription burst and fine-tuning of transcription. |
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| Relations |
| Reanalyzed by |
GSM3282984 |
| SRA |
SRX262233 |
| BioSample |
SAMN02009259 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1117142_nLac.processed.txt.gz |
381.0 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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