|
| Status |
Public on Sep 01, 2012 |
| Title |
control RNAi rep3 |
| Sample type |
SRA |
| |
|
| Source name |
whole day 4 adult worms
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
RNAi: empty vector control RNAi
strain: TJ1060
genotype: fem-15(b26);spe-9(hc88)
tissue: whole worms
|
| Growth protocol |
Three independent biological replicate batches of approximately ~10,000 sterile hermaphrodites of the sterile TJ1060 (fem-15(b26); spe-9(hc88)) strain were grown at the non-permissive (25°C) temperature until the first day of adulthood, at which point half were placed on plates seeded with unc-62 RNAi and half on empty vector RNAi. The worms were then grown for 3 days at 20°C, after which poly-A-purified RNA was isolated
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA was isolated by Trizol extraction followed by phenol-chloroform purification. poly-A-purified RNA was then isolated using the Qiagen Oligotex Mini kit, and 3’ end enriched RNA-seq libraries were prepared in accordance with the 3’SEQ method. 400ng of mRNA was heat sheared for 7.5 minutes at 850C to obtain 200-500bp fragments, and barcoded oligo-dT primers were used to generate cDNA for mRNA fragments located at the 3’ end of transcripts. After second strand synthesis, adaptors were ligated to the 5’ end, and PCR primers (including the barcode sequence) were used in order to enable multiplexed sequencing. The three replicate libraries from worms grown on unc-62 RNAi were then pooled and sequenced in a single flow-cell lane on the Illumina single-end 36bp HiSeq 2000 platform, with the three libraries from worms grown on control RNAi pooled and sequenced in an additional lane.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
3' end enriched poly-A RNA
|
| Data processing |
Basecalls were performed using CASAVA 1.7.0 from Illumina
Reads with 10 or more consecutive A's or T's were removed as likely amplification artifacts, and remaining reads were mapped to the Wormbase reference WS215 genome and transcriptome using Bowtie version 0.12.7, with the options “-a --best --strata -v 2” to obtain all genomic position(s) to which the read mapped with the least number mismatches (up to 2 allowed)
To determine expression for each transcript in the WS215 Wormbase annotation, we counted the number of reads that uniquely mapped to the sense strand (i.e., they did not map equally well elsewhere in the genome).
Genome_build: WS215 (genome + annotated transcripts)
Supplementary_files_format_and_content: Tab-delimited file contains read density (per million uniquely mapped reads) for all annotated WS215 genes.
|
| |
|
| Submission date |
Jul 23, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Eric L Van Nostrand |
| E-mail(s) |
eric.vannostrand@gmail.com
|
| Phone |
6507257611
|
| URL |
http://cmgm.stanford.edu/~kimlab/
|
| Organization name |
Stanford University
|
| Department |
Developmental Biology
|
| Lab |
Stuart Kim
|
| Street address |
279 Campus Drive B341
|
| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
| |
|
| Platform ID |
GPL13657 |
| Series (1) |
| GSE39574 |
RNA-seq quantification of expression changes upon unc-62 knockdown in adult Caenorhabditis elegans. |
|
| Relations |
| SRA |
SRX170369 |
| BioSample |
SAMN01092407 |
