|
| Status |
Public on Apr 04, 2015 |
| Title |
Untreated replicate 1 (GRO-seq) |
| Sample type |
SRA |
| |
|
| Source name |
S2 cell line
|
| Organism |
Drosophila melanogaster |
| Characteristics |
cell type: S2 cells
treatment: Untreated
bru immunoprecipitation: Anti-BrdUTP agarose (Santa Cruz Biotechnology, catalog# sc32323 AC, lot# I2111)
barcode: TCTA
|
| Treatment protocol |
cells untreated or treated with double-stranded RNA against Beta-galactosidase (LacZ-RNAi control) or GAGA factor
|
| Growth protocol |
Cells were grown in M3 media (Sigma) supplemented with bacto-peptone and yeast extract + 10% FBS
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Ten million nuclei were allowed to run-on for 10 minutes at 30°C in a reaction containing ATP, CTP, GTP, and BrUTP. The RNA was isolated using Trizol and acid phenol extractions. The RNAs were base hydrolyzed to an average length of 100-150 nucleotides, and RNAs with incorporated BrU were purified with 2 immunoprecipitations successive using Anti-BrdUTP agarose (Santa Cruz Biotechnology cat# sc32323 AC lot # I2111). The RNA was treated with PNK and polyadenylated using E. coli polyA polymerase to add 50-100 As to their 3’ ends. The polyadenylated RNA was reverse transcribed used with the following bar coded oligonucleotides (INOO3: 5’-pTAGAGATCGTCGGACTGTAGAACTCT-iSp18-CAAGCAGAAGACGGCATACGATTTTTTTTTTTTTTTTTTTTVN, INOO4: 5’-pTGATGATCGTCGGACTGTAGAACTCT-iSp18-CAAGCAGAAGACGGCATACGATTTTTTTTTTTTTTTTTTTTVN). The first 4 bases have the bar-code and iSp18 indicates an 18 carbon spacer between two oligo linkers used for Illumina sequencing. The resulting DNA was gel extracted and circularized using Epicentre CircLigase, and PCR amplified for 12-15 cycles. The cDNA was then sequenced on the Illumina Genome Analyzer.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina Genome Analyzer |
| |
|
| Description |
Nuclear run on with 5-BromoUTP for immunopurification
nascent RNA
|
| Data processing |
Library strategy: GRO-Seq
Barcode removed and read trimmed to 26nt
Adapter removed using cutAdapt with all possible combinations of VN and a threshold of 0.2
Aligned with bowtie to dm3, allowing two mismatches and requiring unique alignment (-m1).
converted bowtie .sam file to the .bed file standard.
normalized per million mapped reads
Genome_build: Release 5 (dm3)
Supplementary_files_format_and_content: bedgraph file; GRO-seq reads normalized to total number of mapped reads (either on the plus or minus strand) after they were trimmed to 26 nucleotides, and mapped to non-repetitive regions of the Drosophila genome using Bowtie.
|
| |
|
| Submission date |
Jul 01, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Nicholas Fuda |
| E-mail(s) |
njf6@cornell.edu
|
| Organization name |
Cornell University
|
| Department |
Molecular Biology and Genetics
|
| Street address |
417 Biotechnology Building, Cornell University
|
| City |
Ithaca |
| State/province |
New York |
| ZIP/Postal code |
14853 |
| Country |
USA |
| |
|
| Platform ID |
GPL9058 |
| Series (2) |
| GSE58955 |
GAGA Factor maintains promoters in nucleosome-free conformation and allows promoter-proximal pausing [GRO-seq] |
| GSE58957 |
GAGA Factor maintains promoters in nucleosome-free conformation and allows promoter-proximal pausing |
|
| Relations |
| BioSample |
SAMN02898405 |
| SRA |
SRX643398 |
