|
| Status |
Public on Aug 03, 2012 |
| Title |
pKF63 SacI |
| Sample type |
SRA |
| |
|
| Source name |
S2 cell culture line
|
| Organism |
Drosophila melanogaster |
| Characteristics |
treatment: pKF63 SacI cut
transfection: pRS425 circular
tag: Firefly luciferase PCR product
cell line: S2
|
| Treatment protocol |
Transfection was performed with Fugene HD (24 ul per 3 ml final culture volume) and 300 ng of each DNA type (see tags above) per 3 ml final volume in 6-well plates. Three days after transfection, the cells were harvested and total RNA was extracted with Trizol.
|
| Growth protocol |
Cells were cultured in standard Schneider's Drosophila medium supplemented with 10% FBS at 25°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using Trizol and size-selected on polyacrylamide/urea gels [~18-30 nt). In a first ligation step, pre-adenylated 3'-linkers were ligated to the 3'-end of the small RNAs (AMP-5'p=5'pCTGTAGGCACCATCAATdideoxyC-3') in the absence of ATP. After gel-purification of the ligation products, a 5'-Adapter was ligated in the presence of ATP 5('-rArCrArCrUrCrUrUrUrCrCrCrUrArCrArCrGrArCrGrC rUrCrUrUrCrCrGrArUrCrU-3'). Following reverse transcription with 5'-ATTGATGGTGCCTACAG-3', PCR was performed to append Adapter sequences for Illumina sequencing as well as indexes for multiplexing. A constant 5'-primer (5'-AATGATACGGCGACCACCGAACACTCTTTCCCTACACGACG-3') was combined with one of four 3'-index primers (5'-CAAGCAGAAGACGGCATACGAGGATCCGATTGATGGTGCCTACAG-3', 5'-CAAGCAGAAGACGGCATACGACAGCTGGATTGATGGTGCCTACAG-3', 5'-CAAGCAGAAGACGGCATACGATCTAGAGATTGATGGTGCCTACAG-3', 5'-CAAGCAGAAGACGGCATACgaATCGATGATTGATGGTGCCTACAG-3'). Sequencing was single-end (either 50, 75 or 78 nt length), 3'-adapters and indexes were sequenced together with the small RNAs.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina Genome Analyzer |
| |
|
| Data processing |
Reads were first sorted according to their sample index, then the 3'-adapter sequence and everything else 3'-of it was removed.
Reads were size-selected for 21-23 nt length.
Reads were sorted into unique sequences and sequence counts => e.g. S1.counts
Genome_build: n/a
|
| |
|
| Submission date |
Jun 27, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Klaus Förstemann |
| E-mail(s) |
foerstemann@lmb.uni-muenchen.de
|
| Organization name |
Ludwig-Maximilians University
|
| Department |
Gene Center
|
| Lab |
Förstemann
|
| Street address |
Feodor-Lynen 25
|
| City |
Munich |
| ZIP/Postal code |
81377 |
| Country |
Germany |
| |
|
| Platform ID |
GPL9058 |
| Series (1) |
| GSE38967 |
A small RNA response to DNA ends in Drosophila |
|
| Relations |
| SRA |
SRX156293 |
| BioSample |
SAMN01081679 |
