|
| Status |
Public on Dec 04, 2008 |
| Title |
GRO-seq rep2 |
| Sample type |
SRA |
| |
|
| Source name |
primary lung fibroblasts
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: IMR90
tissue: lung
antibody: Br-dUTP
|
| Growth protocol |
Cells are maintained in Minimal essential medium (Eagle) with Earle's balance salt solution, 2mM L-glutamine, 1mM sodium pyruvate, 1.5 g/L sodium bicarbonate, 0.1mM non-essential amino acids, and 10% fetal bovine serum. Cells are grown at 37 degrees Celsius at 5% CO2. Cells were harvested at 80% confluency for all assays.
|
| Extracted molecule |
nuclear RNA |
| Extraction protocol |
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.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer |
| |
|
| Description |
Nuclear run on with 5-BromoUTP for immunopurification
Supplementary Raw data files :lib2_s6_tag.txt
lib2_s6_sequence.txt.gz
lib2_s7_tag.txt.gz
lib2_s7_sequence.txt.gz
lib2_s8_tag.txt.gz
lib2_s8_sequence.txt.gz
|
| Data processing |
Reads were aligned with ELAND to the hg18 non-repeat masked reference human genome and alignments to rDNA repeats were then removed.
|
| |
|
| Submission date |
Nov 07, 2008 |
| 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 |
GPL9052 |
| Series (1) |
| GSE13518 |
Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters |
|
| Relations |
| Reanalyzed by |
GSE66031 |
| SRA |
SRX003136 |
| BioSample |
SAMN02195591 |
