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Status |
Public on Oct 01, 2009 |
Title |
drh-3_CIPPNK_seq_s2 |
Sample type |
SRA |
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Source name |
gravid adult worms
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Organism |
Caenorhabditis elegans |
Characteristics |
sample type: gravid adult worms strain: drh-3(ne4253)
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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 |
CIPPNK 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 |
SAMN02196564 |
Supplementary file |
Size |
Download |
File type/resource |
GSM455389_drh-3_CIPPNK_seq_s2.tar.gz |
63.9 Mb |
(ftp)(http) |
TAR |
GSM455389_drh3_CIPPNK_all_1.gff.gz |
9.2 Mb |
(ftp)(http) |
GFF |
Processed data provided as supplementary file |
Raw data provided as supplementary file |
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