|
| Status |
Public on Feb 24, 2010 |
| Title |
small RNAs_alg-3(tm1155);alg-4(ok1041);fog-2(q71) and fog-2(q71)_adult males |
| Sample type |
SRA |
| |
|
| Source name |
Argonaute CSR-1 immunopercipitate
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: alg-3(tm1155);alg-4(ok1041);fog-2(q71) and fog-2(q71)
antibody: anti_CSR-1
|
| Treatment protocol |
n/a
|
| Growth protocol |
For male isolation animals were grown at 20 C for approximately 60h and males were seperated from females to achieve greater than 95% male populations. For spermatid isolations fem-3 worms were grown at 25oC for ~45-50hrs and spermatids were extracted.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Library construction protocol: RNA was extracted from ~200,000 adult worms or from ~200ul isolated spermatids, from alg-3(tm1155);alg-4(ok1041);fog-2(q71), fog-2(q71) male samples and fem-3(q20) purified spermatids. RNA was eluted by extraction with TRI Reagent (MRC Reagents, Inc). RNA was extracted as described in the manufacturer's protocol. Small RNAs from the immunopercipitated complex, and wild type input, were resolved in a 15% polyacrylamide 7M Urea Gel, along with 20 picomoles of RNA standard (18- and 26-nt) in separate lanes. Ethidium Bromide staining was used to visualize the RNA standards. A gel fragment was excised from the sample lanes in the migration range between the two standards. RNA was eluted from the gel fragment in (0.3M NaCl-TE pH7.5) solution overnight and ethanol-precipitated using 20mg of glycogen as the carrier. Gel purified RNA was incubated with 0.05 Unit/µl Tobacco Acid Pyrophosphatase (Epicenter Biotechnologies) in 10µl reaction buffer containing 1 Unit/µl SUPERase Inhibitor (Ambion) for 1h at 37°C. After phenol extraction, the RNA was ethanol precipitated. The gel purified RNA and 1µM of each standard were incubated with 20µM of 3'-end linker, 1 Unit of SuperRNaseIN, 10% DMSO and 3 Units T4 RNA ligase (Takara) in 10µl ligation buffer (50mM Tris-Cl pH7.5, 10mM MgCl2, 6mg/mL BSA, 10mM DTT). The 3' ligated products were gel purified and the RNAs were incubated in the presence of 30µM of 5' adapter oligonucleotide, 1 Unit SuperRNaseIN (Ambion) and 1.5 Units of T4 RNA ligase in ligation buffer (50mM Tris-HCI (pH7.5), 10mM MgCI2, 10mM DTT, 1mM ATP and 10% Dimethyl sulfoxide). The ligated products were gel purified as described above and reverse transcribed in a standard 50µl reaction (SuperScript III, Invitrogen). The cDNA was amplified by PCR and purified in a 10% acrylamide gel. PCR products generated for all the samples were sequenced on a Solexa sequencing platform (Illumina, Inc.).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
18 to 26 nt small RNAs from alg-3/4 males
|
| Data processing |
Small RNA sequences bigger than 17 nt were mapped to the Caenorhabditis elegans genome (WS192, www.wormbase.org). Sequences that matched tRNA or rRNA sequences were then removed. Only full-length complete matches were allowed. siRNA-targeted loci were defined as a region containing at least 25 reads per million matching reads. Relative abundance at each locus was determined by calculating (22G-RNAs in mutant(or)IP)/(22G-RNAs in wt+22G-RNAs in mutant(or)IP ).
|
| |
|
| Submission date |
Oct 26, 2009 |
| Last update date |
May 15, 2019 |
| Contact name |
Craig Mello |
| Organization name |
University of Massachusetts Medical School
|
| Department |
Program in Molecular Medicine
|
| Lab |
Craig Mello
|
| Street address |
368 Plantatoin Street, Suite AS5-2047
|
| City |
Worcester |
| State/province |
MA |
| ZIP/Postal code |
01605 |
| Country |
USA |
| |
|
| Platform ID |
GPL9269 |
| Series (2) |
| GSE18729 |
High throughput sequencing of endogenous small RNAs from AGO pathway mutants |
| GSE18731 |
AGO pathway |
|
| Relations |
| SRA |
SRX017288 |
| BioSample |
SAMN00009556 |
