 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Oct 12, 2012 |
| Title |
NELF_RNAi GRO-seq 1+2 |
| Sample type |
SRA |
| |
|
| Source name |
NELF_RNAi GRO-seq
|
| Organism |
Drosophila melanogaster |
| Characteristics |
cell line: Schneider Line 2 (S2)
antibody: anti-BrdU (Santa Cruz Biotech (sc-32323-AC))
treatment: NELF_RNAi
|
| Treatment protocol |
Cells that were RNAi treated received dsRNA targeting the NELF-B and NELF-E subunits or “mock” dsRNA against b-galactosidase for 96 hours prior to harvesting cells.
|
| Growth protocol |
S2 cells were maintainedat 25oC in M3+BPYE media supplemented with 10% FBS
|
| Extracted molecule |
nuclear RNA |
| Extraction protocol |
Libraries untr, Mock and NELF RNAi: Nuclei isolation and run-on reactions are performed using standard protocols with the exception that 5-Bromo-UTP is used in place UTP, and the concentration of CTP is adjusted to 1µM. Samples are treated with Rnase-free RQ1 DNase (promega) and Proteinase K (invitrogen). RNA is then extracted twice with Leder phenol and once with chloroform, then precipitated. Isolated RNA is then base hydrolyzed to the desired size and RNA fragments are then isolated by binding to anti-deoxy-BrU beads, washed several times, and eluted from the beads. The RNAs are treated at low pH with tobacco acid pyrophosphatase and then are treated at low pH with T4 polynucleotide kinase (PNK). The pH is then raised and the RNA is treated again with PNK, except now in the presence of ATP. A standard Illumina adapter is then added to the 5’-end with T4-RNA ligase and the RNA is bound to anti-deoxy-BrU beads. This process is then repeated for the addition of the Illumina 3’-adapter. The affinity-enriched RNAs are then reverse transcribed, amplified, and PAGE purified. This cDNA was then sequenced on the Illumina Genome Analyzer. Libraries Plus Sark and Minus Sark: Plus- and minus-Sarkosyl matched GRO-seq libraries were made with three sequential bead enrichment steps as above, but a RNA cloning strategy developed by Ingolia et al. (Science, 2009, Vol. 324. no. 5924, pp. 218 - 223), was used to prepare the samples for sequencing with the following modifications. PNK treatment to remove 3’-phosphates was performed after the first bead enrichment. cDNAs were not PAGE purified after circularization because the range of sizes (~150-350bp) of the cDNA prevented efficient separation of the circularized and linearized cDNAs. Samples were amplified and PAGE purified before sequencing on the Illumina Genome Analyzer.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina Genome Analyzer |
| |
|
| Description |
Nuclear Run-on (nascent RNA). Two replicate experiments combined.
|
| Data processing |
Reads were trimmed to 26bp and aligned to Drosophila melanogaster (Dm3 version) genome with MAQ. The number of reads at each position were then normalized for each library by dividing by the number of reads (in millions) that mapped uniquely to the genome, to a representative rRNA locus, and to representative tRNA. This accounts for the total transcription in cells after treatments that will differentially affect transcription by one or more of the RNA polymerases (I, II, or III).
|
| |
|
| Submission date |
Aug 10, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
Leighton James Core |
| E-mail(s) |
ljc37@cornell.edu
|
| Organization name |
Cornell University
|
| Department |
Moleular Biology and Genetics
|
| Lab |
John T. Lis
|
| Street address |
417 Biotechnology Building
|
| City |
Ithaca |
| State/province |
NY |
| ZIP/Postal code |
14853 |
| Country |
USA |
| |
|
| Platform ID |
GPL9058 |
| Series (2) |
| GSE23543 |
GRO-seq in Drosophila melanogaster S2 cells |
| GSE23544 |
Comparison of GRO-seq with Pol II (Rpb3) ChIP-seq in Drosophila melanogaster S2 cells |
|
| Relations |
| Reanalyzed by |
GSM3274628 |
| Reanalyzed by |
GSM3274633 |
| SRA |
SRX026305 |
| SRA |
SRX026393 |
| BioSample |
SAMN00109976 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM577246_NELF1and2_CORE_Normed_Minus.bed.gz |
24.8 Mb |
(ftp)(http) |
BED |
| GSM577246_NELF1and2_CORE_Normed_Plus.bed.gz |
23.3 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
|
|
|
|
 |
