 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Aug 12, 2016 |
| Title |
rrp6D_BMA64_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
yeast cultures
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain genotype: MATa; ura3-1; trp1Δ 2; leu2-3,112; his3-11,15; ade2-1; can1-100 rrp6Δ::hphMX4
genetic background: BMA64
mating type: alpha
|
| Treatment protocol |
For anchor away experiments, WT HHY168 and FRB-tagged strains were treated for one hour with 0.1% v/v 1 mg/mL rapamycin (in 90% ethanol, 10% Tween-20) to yield a final concentration of 1 μg/mL.
|
| Growth protocol |
cultures were grown in YPD (1% yeast extract, 2% bacto-peptone, 2% dextrose) at 30°C at 200 rpm and harvested at OD 0.4 to 0.6
|
| Extracted molecule |
total RNA |
| Extraction protocol |
All cultures were harvested by centrifugation at 4000 rpm for 2 minutes, washed in deionized water, and spun down in microcentrifuge tubes. The supernatant was removed and pellets were flash-frozen in liquid nitrogen and stored at -80°C. RNA was extracted by standard phenol/chloroform extraction and DNase I-treated.
For the WT and rrp6Δ samples, libraries were prepared per the manufacturer’s protocol. For all other samples, the reverse transcription (RT) was performed at 42°C instead of 37°C to minimize imperfect priming between the oligo-dT primer and internal A-rich sequences. After denaturation and annealing of the RNA and the RT primer, the samples were kept on the heating block while 42°C pre-warmed RT enzyme solution was added to each sample. For samples with RT performed at 42°C, 15 PCR cycles instead of 12 were performed to compensate for decreased RT yields.
A subset of samples (labeled "ePAP") were treated with E.coli poly(A) polymerase in vitro prior to library preparation. 10 ug of total RNA was treated with ATP and E.coli poly(A) polymerase in a total volume of 20 ul at 37°C for 30 minutes, according to manufacturer’s recommendations (NEB #M0276L). After phenol/chloroform extraction and ethanol precipitation, 5 ug of RNA was subjected to rRNA depletion with Ribo-Zero Gold rRNA Removal Kit (Illumina, Inc.), prior to 3´-end poly(A)+ library preparation.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
RNA extracted from yeast cultures and Dnase I-treated
|
| Data processing |
Reads were trimmed with TrimGalore v0.4.0 requiring a minimum final length of 15 nucleotides.
Trimmed reads were mapped using RNA-STAR v2.4.0k to the R64-2-1 version of the S.cerevisiae genome obtained from the Saccharomyces genome database (SGD) using default mapping parameters and allowing only uniquely mapped reads (Dobin et al., 2013).
5´ ends of reads at each chromosomal coordinate were aggregated to generate bedgraphs with the bedtools v2.25.0 genomecov function.
Mapped positions were first filtered for A/G richness in the immediate genomic region downstream. Poly(A) sites with six genomically encoded A nucleotides downstream (with up to two G nucleotides) were flagged as potential internal oligo-dT mis-priming events and excluded from the analysis.
Analysis of the most abundant internal priming events by-passing this filter revealed that A-richness in across an 18 bp region explained internal priming events not flagged by the 6 bp filter. Therefore, as an additional stringency filter, we excluded poly(A)-sites with 12 or more adenosines present in these downstream 18 nt. On the order of ~1% of sequencing reads involving long stretches of T’s, possibly due to priming internal to the poly(A) tail or due to a low level of contamination of the sequencer with Illumina Read 1 primer. As an additional quality control step, poly(A) sites were flagged if the preceding 15 or more nucleotides were adenosines.
The total reads for each bedgraph (both strands combined) remaining after filtering steps were normalized to 1 million.
Genome_build: R64-2-1 version of the S288C genome of S.cerevisiae (Saccharomyces genome database)
Supplementary_files_format_and_content: bedgraph with each strand separate
|
| |
|
| Submission date |
Aug 11, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Kevin Roy |
| E-mail(s) |
[email protected]
|
| Organization name |
UCLA
|
| Department |
Chemistry and Biochemistry
|
| Lab |
Guillaume Chanfreau
|
| Street address |
607 Charles E. Young Drive East
|
| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90095 |
| Country |
USA |
| |
|
| Platform ID |
GPL13821 |
| Series (2) |
| GSE75586 |
3´-end sequencing of poly(A)+ RNA in wild-type Saccharomyces cerevisiae and nuclear exosome mutant strains |
| GSE75587 |
Nuclear exosome |
|
| Relations |
| BioSample |
SAMN05560706 |
| SRA |
SRX2013601 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2268787_rrp6D_BMA64_rep1_minus_strand.bedgraph.gz |
1.2 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM2268787_rrp6D_BMA64_rep1_plus_strand.bedgraph.gz |
1.2 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
