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Sample GSM455401 Query DataSets for GSM455401
Status Public on Oct 01, 2009
Title smg5_CIPPNK_seq_prb_s5
Sample type SRA
 
Source name gravid adult worms
Organism Caenorhabditis elegans
Characteristics sample type: gravid adult worms
strain: smg-5(r860)
Growth protocol animals were grown at 20 C for approximately 60h.
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.)
 
Library strategy ncRNA-Seq
Library source transcriptomic
Library selection size fractionation
Instrument model Illumina Genome Analyzer II
 
Description CIPPNK
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 ).
 
Submission date Sep 22, 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)
GSE18215 High throughput sequencing of mutants in the WAGO pathway
GSE18231 WAGO pathway
Relations
SRA SRX013292
BioSample SAMN00005374

Supplementary file Size Download File type/resource
GSM455401_smg5_CIPPNK.gff.gz 32.2 Mb (ftp)(http) GFF
SRA Run SelectorHelp
Processed data provided as supplementary file
Raw data are available in SRA

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