|
| Status |
Public on Jan 01, 2017 |
| Title |
mRNA-seq_met-2set-25_OP50 |
| Sample type |
SRA |
| |
|
| Source name |
Young adult whole animal
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
genotype/variation: met-2(n4256);set-25(ok5021)
temperature: 19˚C
feeding: OP50
|
| Growth protocol |
C. elegans strains N2, hrde-1 (tm1200), set-32(ok1457), met-2(n4256);set-25(ok5021), and met-2(n4256);set-25(ok5021);set-32(ok1457) were cultured on NGM plates with E. coli OP50 as the food source in a temperature controlled incubator at 19°C. Synchronized worms were raised at 19°C and young adult worm samples were collected.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was extracted from frozen synchronized adult worm grinds (100-200 μl, pulverized in liquid nitrogen by grinding) using Trizol reagent (Life Technologies). mRNA was enriched using Poly(A)Purist MAG kit (Life Technologies).
0.5-1 μg of mRNA was used for each mRNA-seq library. DNA was removed from the RNA sample by DNase I treatment according to manufacture’s instruction (New England Biolabs). RNA was fragmented by hydrolysis using Fragmentation Reagent Kit (Ambion). RNA was treated with T4 PNK (NEB). Then RNA 3’ end was ligated to IDT linker-1 (5’-rAppCTGTAGGCACCATCAATC-3’) using T4 RNA ligase 2, truncated (NEB, M0242S). RNA library of about 35-60 nt was size selected using 8 % polyacrylamide (acrylamide:bis-acrylamide = 19:1) gels containing 8 M urea (size selection also eliminated excess IDT linker-1). RNA 5’end was ligated to DNA-RNA hybrid oligo (5’-ACGCTCTTCCGATCTrNrNrNrN-3’, where rNrNrNrN is a 4-nt barcode) using T4 RNA ligase 1 (NEB) with ATP. First strand cDNA was synthesized using SuperScript III RT kit (Life Technologies) and primer SG-460 (5’-GGAGTTCAGACGTGTGCTCTTCCGATCTATTGATGGTGCCTACAG-3’). cDNA library was PCR amplified at early cycle (tested by different cycle number to capture linear amplification phase and avoid over amplification). PCR was done using a common primer SG-465 (5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’) and primer with 6-nt index for multiplexing (5’-CAAGCAGAAGACGGCATACGAGAT[ index]GTGACTGGAGTTCAGACGTGTGCTCTTCC-3’). Amplified library was size selected by 2% agarose gel and gel purified using QIAquick Gel Extraction Kit (QIAGEN).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
The raw reads were processed by trimming 4-nt inline barcode at the 5' end and linker sequence from the 3' end
The processed reads were aligned to the C. elegans annoated gene database (spliced) using bowtie[0.12.7]
The mRNA expression level for each gene was calculated by counting the number of reads that were aligned to the sense strand of the gene. One pseudo-count was added to each gene to avoid zero counts. The counts were normalized by the sequencing depth (in millions) for each sample.
Genome_build: WS190/ce6
Supplementary_files_format_and_content: .xlsx file containing the mRNA expression levels (rpkm) in all 5 samples for all annotated C. elegans genes. Number correspond to the total 100%-matching reads aligned in sense orientation to the mRNA and normalized by mRNA length and total sequencing depth in million. Additional columns define germline expression (oocyte only, sperm only or germline (both) = intrinsic) and germline nuclear RNAi target type: heterochromatin (GHR) or transcriptional silencing (GRTS).
|
| |
|
| Submission date |
Sep 07, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Sam Guoping Gu |
| E-mail(s) |
ggu@dls.rutgers.edu
|
| Organization name |
Rutgers University
|
| Department |
Molecular Biology and Biochemistry
|
| Street address |
604 Allison Road
|
| City |
Piscataway |
| State/province |
New Jersey |
| ZIP/Postal code |
08854 |
| Country |
USA |
| |
|
| Platform ID |
GPL13657 |
| Series (2) |
| GSE86515 |
Decoupling the downstream effects of germline nuclear RNAi reveals that transcriptional repression and heritable RNAi are independent of the H3K9me3 response in C. elegans [RNA-seq] |
| GSE86517 |
Decoupling the downstream effects of germline nuclear RNAi reveals that transcriptional repression and heritable RNAi are independent of the H3K9me3 response in C. elegans |
|
| Relations |
| BioSample |
SAMN05736286 |
| SRA |
SRX2144197 |
