|
| Status |
Public on Aug 01, 2013 |
| Title |
Δelp3 RNA-seq repeat |
| Sample type |
SRA |
| |
|
| Source name |
YWG382
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
poly-a selection: oligo-dT cellulose
triton addition: After lysis
strain: BY4741
|
| Treatment protocol |
Cells were treated with 100ug/ml cycloheximide, then harvested by centrifugation. Cell pellet was frozen in liquid nitrogen.
|
| Growth protocol |
Yeast were grown to mid-exponential phase (OD~0.7) at 30C in YPD
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Small RNA sequencing by polyadenylation and circularization. RNA fragments were dephosphorylated, purified by filtration then gel to isolate ~28nt fragments. After subtractive hybridization to remove rRNA, fragments were poly(A) tailed and reverse transcribed with an anchored oligo-dT primer, followed by cDNA circularization and PCR. Frozen cell pellets were thawed in lysis buffer and lysis was performed with glass beads as described in the text. Extracts were clarified by a 20' spin at 11,500 rpm in an SS-34 rotor.
Footprints: 50 OD units of extract were treated with 150U of RNAse1 and subsequently purified from the monosome region of a sucrose gradient. RNA was extracted by hot acid phenol. Total RNA: RNA was extracted from 50 OD of extract by SDS and hot acid phenol, and poly(A) RNA was purified on oligo-dT cellulose or DynaBeads. RNA was fragmented at 95C with zinc chloride
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
Small RNA sequencing by polyadenylation and circularization. RNA fragments were dephosphorylated, purified by filtration then gel to isolate ~28nt fragments. Fragments were poly(A) tailed and reverse transcribed with an anchored oligo-dT primer, followed by cDNA circularization and PCR
|
| Data processing |
align first 21nt of reads to rRNA, with up to 3 mismathces allowed, discarding any reads that map
align first 21nt of remaining reads to entire yeast genome, allowing up to 3 mismatches
align remaining unmapped reads to splice junction sequences, and merge with genome mapping
Reads whose 5' ends map from 12nt 3' of CDS start to 14nt 5' of CDS end are assigned to that gene (first 8 codons are ommitted to avoid artifacts due to the use of cylcoheximide in library preparation)
RPKMs are computed as reads per kilobase of CDS sequence per million reads mapping to all CDSs
Genome_build: Saccharomyces cerevisiae May 26 2010
Supplementary_files_format_and_content: tab-delimited tables of reads and RPKMs for all genes and samples. Technical replicates for RNA-seq are pooled.
|
| |
|
| Submission date |
Mar 20, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Boris Zinshteyn |
| E-mail(s) |
zinshteyn.boris@gmail.com
|
| Organization name |
Johns Hopkins University School of Medicine
|
| Department |
Molecular Biology and Genetics
|
| Lab |
Green
|
| Street address |
725 N Wolfe St, PCTB 714
|
| City |
Baltimore |
| State/province |
Maryland |
| ZIP/Postal code |
21205 |
| Country |
USA |
| |
|
| Platform ID |
GPL13272 |
| Series (1) |
| GSE45366 |
Loss of a Conserved tRNA Anticodon Modification Perturbs Cellular Signaling |
|
| Relations |
| SRA |
SRX252853 |
| BioSample |
SAMN01985053 |
