|
| Status |
Public on Dec 19, 2013 |
| Title |
WT males 20°C small RNA |
| Sample type |
SRA |
| |
|
| Source name |
fog-2(q71) males 20°C
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
barcode: 5' barcode (A) TGAC
barcode: 3' barcode (1) CTGTAGGCACCATCAAT
genotype/variation: WT
culture conditions: 20°C
|
| Treatment protocol |
Small RNAs of 10-40nt from total RNAs were resolved on a 15% polyacrylamide 7M Urea Gel, along with 2 picomoles of RNA standard (18- and 40-nt) in separate lanes. SYBR gold 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 isopropanol-precipitated with 20mg of glycogen as the carrier. The gel purified RNA was incubated with 0.25 U/µl TAP (Epicentre) and 1U/µl SuperRNaseIN (Ambion) in 10µl reaction containing 1X buffer at 37 oC for 1 hr, followed by phenol-chloroform extraction and isopropanol precipitation. The TAP-treated samples were then incubated with 5µM of 3’-end linker (1, 2, 3 from IDT DNA Technologies), 1 Unit/µl of SuperRNaseIN, 10% DMSO and 1 U/µl T4 RNA ligase (Takara) in 10µl ligation buffer (50mM Tris-Cl pH7.5, 10mM MgCl2,10mg/mL BSA, 10mM DTT). The 3’ ligated products were gel purified, eluted, precipitated and then incubated with 5 µM of 5’ adapter oligonucleotide with 4nt barcode at the 5' end, 1 Unit/µl SuperRNaseIN (Ambion) and 2 U/µl of T4 RNA ligase in ligation buffer (50mM Tris-HCI pH7.5), 10mM MgCI2, 10mM DTT, 1mM ATP) and 10% DMSO. The ligated products were gel purified and reverse transcribed in a standard 20µl reaction (SuperScript III, Invitrogen). The cDNA was amplified by PCR and purified in a 8% acrylamide gel. PCR products generated for all the samples were sequenced on a Solexa sequencing HiSeq platform (Illumina, Inc.) and single-end 50 nt reads were obtained.
|
| Growth protocol |
C. elegans worm grown at 20°C and 25°C
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Small RNA cloning, 5' and 3' ligation dependent
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
Sample name: fog220
WT males 20°C small RNA
small RNA profiling
|
| Data processing |
Remove 5' barcode and 3' barcode (or called linker)
Map the reads to WS215 genome and annotation
A custom PERL pipeline is used to summarize reads and generate histogram at single nt level.
Generic genome browse is used to visualize the data
Genome_build: WBcel215 (WormBase WS215)
Supplementary_files_format_and_content: txt format table for sequence and read number mapping the genome
|
| |
|
| Submission date |
Aug 08, 2013 |
| 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 |
GPL13776 |
| Series (1) |
| GSE49672 |
Argonautes promote male fertility and provide a paternal memory of germline gene expression in C. elegans |
|
| Relations |
| BioSample |
SAMN02314090 |
| SRA |
SRX333002 |
