|
| Status |
Public on Jan 13, 2010 |
| Title |
rrf-3 male 1 (barcode: TATG) |
| Sample type |
SRA |
| |
|
| Source name |
whole males
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
tissue: whole organism
genotype: rrf-3(pk1426); him-8(e1489)
strain: PD3330
|
| Growth protocol |
All animals were raised to young adult stage at 25°C on solid media with E. coli (OP50) as food.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Libraries were prepared using a protocol similar to miRNA cloning methods in which small RNA species are flanked between adapter sequences to allow for PCR and sequencing. Unlike miRNA capture methods, this protocol does not select for small RNAs with 5' monophosphates and hence captures mainly the secondary-type, triphosphorylated endo siRNAs. The small RNA was extracted from frozen tissue using the mirVana miRNA Isolation kits.To remove the bias for 5' monophosphorylated species but preserve the bias towards 3' hydroxylated species, we treated the small RNAs with Antarctic phosphatase following addition of the 3' adapter with T4 RNA ligase 1. The resulting hydroxylated 5' ends were phosphorylated by treatment with T4 polynucleotide kinase and ATP in order to form a subtrate for T4 RNA ligase 1-mediated addition of the 5' adapter. After ligation of both the 3' and 5' adapters, the adapters were extended during the reverse transcription and PCR steps to include complete primer binding sites for subsequent amplification and sequencing reactions. To protect against cross-contamination and to allow for pooling of samples, we modified the 5' adapter sequence to include the Illumina genomic sequencing primer immediately followed by a 4-nt barcode.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Data processing |
The parsed reads correspond to the RNA captured between the 3' and 5' adapters. The 3'-adapter sequences were trimmed off by scanning from the 3' end of the 36-nt reads for the first instance of the first 4 nt of the adapter: CTGT. Reads that lacked CTGT in the 3'-most 13 nt were excluded from further analysis. After barcode sorting, the 4 nt of barcode were trimmed off the 5' ends to yield 19- to 28-nt reads corresponding to the captured small RNAs.
The alignments contain genomic alignments produced from the parsed reads and can be uploaded for viewing on the UCSC genome browser. A local installation of BLAT was used with the C. elegans WS190 genome sequence as a reference. The BLAT parameters were set to default except -tileSize=10 and -stepSize=5.
|
| |
|
| Submission date |
Oct 06, 2009 |
| Last update date |
Jun 11, 2013 |
| Contact name |
Sam Guoping Gu |
| E-mail(s) |
ggu@dls.rutgers.edu
|
| Organization name |
Rutgers University
|
| Department |
Molecular Biology and Biochemistry
|
| Lab |
Sam Gu
|
| Street address |
Nelson Labs A125, 604 Allison Road
|
| City |
Piscataway |
| State/province |
NJ |
| ZIP/Postal code |
08854 |
| Country |
USA |
| |
|
| Platform ID |
GPL9269 |
| Series (1) |
| GSE18429 |
Endo-siRNAs in C. elegans spermatogenesis and their requirement for the RdRP RRF-3 |
|
| Relations |
| BioSample |
SAMN02196601 |
