|
| Status |
Public on Sep 25, 2011 |
| Title |
3' RNA-seq Watson strand WT cells_and_3' RNA-seq Crick strand WT cells |
| Sample type |
SRA |
| |
|
| Source name |
yeast poly A+ mRNA
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genotype/variation: W3031B
growth conditions: cells were grown in SC 2%raffinose+galactose to O.D. ~ 0.2 at 25º then shifted to SC 2% glucose for 8 hr
|
| Growth protocol |
cells were grown in SC 2%raffinose+galactose to O.D. ~ 0.2 at 25º then shifted to SC 2% glucose for 8 hr
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
3' end cDNA library construction
Oligo-dT primed libraries of 3' ends were made as described (Mangone, M. et al. Science 329, 432-435, 2010) with modifications outlinedbelow and sequenced with the Illumina Genome Analyzer IIx. Single-end 25 base reads (after removing oligodT(12) and barcodes) were mapped to the UCSC yeast genome (sacCer2, SGD genome Jun 2008 assembly) with Bowtie version 0.12.5.
First-strand cDNA synthesis (20µl final volume) was with SuperScript II RT (Invitrogen) at 42º according to the manufacturer's instructions. Poly(A)+ RNA (200-400ng) purified from WT (W3031B) or Yra1-depleted (DBY1276-2) cells using BioMag Oligo (dT)20 particles (Polysciences, Inc.) was primed with the following biotinylated oligo at 0.5mg/ml:
5'-biotin-ACACTCTTTCCCTACACGACGCTCTTCCGATCTXXTTTTTTTTTTTTVN-3' where XX represents a barcode (CG or GC). The second strand (100µl final volume) was synthesized using DNA polymerase I (30U, Fermentas) in the presence of RNaseH (1U, Fisher Scientific) at 15 'C for 2.5 hours and purified using a QIAquick column (Qiagen). cDNA was digested with NlaIII, purified on a QIAquick column and immobilized on 25µl of Streptavidin Dynabeads M-280 (Invitrogen) in B&W buffer (5mM Tris, pH7.5, 0.5mM EDTA, 1M NaCl). Streptavidin-bound cDNA was washed twice with B&W buffer and twice with of 10mM Tris, pH 8 (500µl each wash), and ligated to 5µl (50 pmols) annealed NlaIII-linkers (top: 5'-5phos-CATGGGAAGAGCGGTTCAGCAGGAATGCCGAG-3'; bottom: 5'-CTCGGCATTCCTGCTGAACCGCTCTTCC-3') using T4 DNA ligase (5U, Fermentas) overnight at 16'C (30µl final volume). The beads were washed at room temperature twice with B&W buffer and twice with TE (500µl each wash). Beads were resuspended in 200µl TE and cDNA was eluted by incubation with 200µl phenol/chloroform for 30 minutes at 65'C with frequent vortexing. 3'-cDNA libraries were purified using a QIAquick column and PCR-amplified (18 cycles) using Phusion polymerase (NEB) using Illumina paired end primers.
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| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
oligo-dT primed cDNA
|
| Data processing |
Single-end 25 base reads (after removing oligodT(12) and barcodes) were mapped to the UCSC yeast genome (sacCer2, SGD genome Jun 2008 assembly) with Bowtie version 0.12.5. We generated bedGraph profiles representing the number of aligned reads at each genomic position (bp). The mapping process was performed in two steps to eliminate artifacts due to internal priming at runs of A's. First, we aligned the reads after removing the seven nucleotides only at the 5? end (three nucleotide barcode and the four oligo dT). These mapped reads were discarded as artefacts due to internal priming at runs of As. In the second step, we realigned the reads unmapped in the first step after removing the 15 nucleotides at the 5? end comprising the barcode and T12).
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| |
|
| Submission date |
Jul 15, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
David L. Bentley |
| E-mail(s) |
david.bentley@ucdenver.edu
|
| Organization name |
U. Colorado School of Medicine
|
| Department |
Biochemistry and Mol. Genetics
|
| Street address |
12801 E. 17th Ave
|
| City |
Aurora |
| State/province |
CO |
| ZIP/Postal code |
80045 |
| Country |
USA |
| |
|
| Platform ID |
GPL13272 |
| Series (2) |
| GSE30703 |
Budding yeast mRNA poly(A) site mapping |
| GSE30706 |
Budding yeast mRNA export and processing factors |
|
| Relations |
| SRA |
SRX084585 |
| BioSample |
SAMN00672590 |
