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| Status |
Public on Oct 01, 2009 |
| Title |
glp-4_Terminator_seq_s3 |
| Sample type |
SRA |
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| Source name |
adult worms lacking germline
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| Organism |
Caenorhabditis elegans |
| Characteristics |
sample type: adult worms lacking germline
strain: glp-4(bn2)
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| Growth protocol |
animals were grown at 20 C for approximately 60h.
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| Extracted molecule |
total RNA |
| Extraction protocol |
CIP-PNK protocol: 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 |
ncRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
Terminator
tar file of Illumina *_seq.txt files provided as supplementary file
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| Data processing |
Small RNA sequences longer 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 perfect 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 22, 2009 |
| Last update date |
May 30, 2014 |
| Contact name |
Craig Mello |
| 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 |
368 Plantatoin Street, Suite AS5-2047
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| City |
Worcester |
| State/province |
MA |
| ZIP/Postal code |
01605 |
| Country |
USA |
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| Platform ID |
GPL9269 |
| Series (2) |
| GSE18215 |
High throughput sequencing of mutants in the WAGO pathway |
| GSE18231 |
WAGO pathway |
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| Relations |
| BioSample |
SAMN02196599 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM455394_glp-4_Terminator_seq_s3.tar.gz |
59.5 Mb |
(ftp)(http) |
TAR |
| GSM455394_glp4_terminator.gff.gz |
6.0 Mb |
(ftp)(http) |
GFF |
| Processed data provided as supplementary file |
| Raw data provided as supplementary file |
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