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Sample GSM977585

Query DataSets for GSM977585

Status

Public on Sep 09, 2012

Title

30°C_biological_replicate1

Sample type

SRA

Source name

polyA RNA, RNase V1 at 30°C

Organism

Saccharomyces cerevisiae

Characteristics

strain: S288C
growth phase: log
folding and cutting temperature: 30°C

Treatment protocol

PolyA-isolated RNA was folded and structure probed at 23°C, 30°C, 37°C, 55°C and 75°C.

Growth protocol

Yeast strain S288C was grown at 30°C to log phase (~4x10^7 cells/ml) in YPD medium.

Extracted molecule

polyA RNA

Extraction protocol

RNA was isolated from yeast cells using the Poly(A)Purist MAG Kit (Life Technologies). The cleavage sites were captured and cloned using SOLiD™ Small RNA Expression Kit (Ambion).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

AB SOLiD System 2.0

Description

H3
cDNA
RNA structure probing and deep sequencing of double-stranded regions in the yeast transcriptome at 30°C-replicate1.

Data processing

The yeast genome was downloaded from the Saccharomyces Genome Database (SGD, http://www.yeastgenome.org). The yeast transcriptome was assembled using SGD annotations (downloaded June 2008). The length of the untranslated regions (UTRs) for each transcript was taken from Nagalakshmi et al., 2008 (PMID 18451266).
Raw reads were mapped to the yeast transcriptome using SHRiMP2.
V1 reads were normalized to the library size of each sequencing lane. This size could be simply estimated by the total mapped reads of that lane. But this estimate can be severely biased (Bullard et al., 2010; Li, J., Witten, D. M., Johnstone, I.,Tibshirani, R., 2011; Robinson and Oshlack, 2010) as reads at melted bases are dispersed to other bases. To reduce this bias, we instead applied PoissonSeq (Li, J., Witten, D. M., Johnstone, I., Tibshirani, R., 2012 (PMID 22003245)), which makes an estimate of those bases that are likely to be stable under different temperatures based on the Poisson goodness-of-fit statistic and then estimates the sizes using reads of these bases. Since the inclusion of positions with few reads lowers the accuracy of PoissonSeq, the data that we input into PoissonSeq was not reads on all bases, but only at bases that are peaks in both duplicated libraries of at least one of the five temperatures. In each library, a peak was defined as a base whose number of reads is larger than (1) the base on its left and the base on its right, (2) the average of bases on the same gene, and (3) the average of all bases. In total, 231,068 bases were selected. Using these estimated library sizes, we divided counts from each sample by the corresponding size. The log2 of these normalized reads were then treated as the V1 scores.
Genome_build: June 2008 (SGD/sacCer2)
Supplementary_files_format_and_content: Tab-delimited text file (GSE39680_processed.txt) with genes and their base readings for each sample. This file is linked to the Series record as a supplementary file.

Submission date

Jul 26, 2012

Last update date

May 15, 2019

Contact name

Kun Qu

E-mail(s)

kqu@stanford.edu

Organization name

Stanford University

Department

Dermatology