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| Status |
Public on Oct 02, 2009 |
| Title |
small RNAs from ego-1 mutant animals |
| Sample type |
SRA |
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| Source name |
ego-1(om97) adult animals
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| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: ego-1(om97)
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| Growth protocol |
animals were grown at 20 C for approximately 60h. Csr-1(tm892), ego-1(om97) and DA1316 animals were counter selected on ivermectin
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| Extracted molecule |
total RNA |
| Extraction protocol |
[CSR-1 IP samples] RNA was eluted from CSR-1 immunopercipitated complex 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.)
[csr-1(tm892), ego-1(om97) and DA1316 samples] RNA was 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 treated with 1 Unit/µl of Alkaline Phosphatase, Calf Intestine (NEB) in 100mM NaCl, 50mM Tris-HCl, 10mM MgCl2, 1mM Dithiothreitol, pH 7.9 at 25°C and 1 Unit/µl SuperRNaseIn (Ambion) for 1 hour at 37 °C. After phenol extraction, 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 treated with 1 Unit/µl Polynucleotide Kinase in 1x Polynucleotide Kinase buffer (70mM Tris-HCl, 10mM MgCl2, 5mM Dithiothreitol, pH 7.6 at 25°C), 2mM ATP, 1 Unit/µl SuperRNAseIN. After phenol extraction, 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.)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
small RNAs, 18 to 26 nt
18 to 26 nt small RNAs from ego-1 mutant animals
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| 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 ).
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| Submission date |
Sep 18, 2009 |
| Last update date |
May 15, 2019 |
| Contact name |
Julie M. Claycomb |
| Organization name |
University of Massachusetts Medical School
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| Department |
Program in Molecular Medicine
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| Lab |
Craig Mello
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| Street address |
373 Plantation Street Biotech2 Suite 219
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| City |
Worcester |
| State/province |
MA |
| ZIP/Postal code |
01605 |
| Country |
USA |
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| Platform ID |
GPL9269 |
| Series (2) |
| GSE18165 |
High-throughput pyrosequencing of endogenous small RNAs from CSR-1 IP complexes and csr-1(tm892) and ego-1(om97) mutants |
| GSE18167 |
Gene expression in csr-1 mutants and sequencing of sRNAs from CSR-1 IP complexes and csr-1 and ego-1 mutants |
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| Relations |
| SRA |
SRX013298 |
| BioSample |
SAMN00005380 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM454003_ego1om97_17up_geex_normed.txt.gz |
24.0 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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