GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM558929

Query DataSets for GSM558929

Status

Public on Aug 24, 2010

Title

Total RNA from yeast, treated with RNase S1, replicate 2

Sample type

SRA

Source name

Yeast polyA RNA, RNase S1-treated

Organism

Saccharomyces cerevisiae

Characteristics

protocol: RNase S1

Growth protocol

S cerevisiae is grown to log phase at 23C, 200rpm.

Extracted molecule

polyA RNA

Extraction protocol

Total RNA is extracted from log phase yeast using hot acid phenol, chlorofom and ethanol precipitation. The RNA is then double polyA selected and folded in vitro in structure buffer(10mM Tris pH7, 10mM Mg2+, 100mM KCl). The folded RNA is cleaved using RNase V1 or S1 nuclease for 15min at room temperature. The enzymes are inactivated by phenol chloroform extraction. The RNA is then fragmented to a size of around 200 bases using alkaline hydrolysis (50mM Sodium Carbonate [NaHCO3/Na2Co3] pH 9.2, 1 mM EDTA) at 95C for 3min. The RNA is ran onto a 6% PAGE gel and fragments between 50-200 bases are cut out and eluted out of the gel. The RNA fragments are then ligated to 5' and 3' adapters and reverse transcribed. This is followed by PCR (18-20cycles) and another PAGE to size select for a library size of 150-250 bases.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

AB SOLiD System

Description

S1 rep. 2
PolyA transcripts from log-phase growing yeast, treated with structure-specific enzyme.

Data processing

Obtained sequences were truncated to 35bp before mapping, and required to map uniquely to either the yeast genome or transcriptome, allowing up to one mismatch and no insertions or deletions. Mapping of the short reads to the yeast transcriptome was done using version 1.1.0 of SHRiMP, which supports direct "color space" mapping, downloaded from http://compbio.cs.toronto.edu/shrimp/. We required the alignment to start from the first base of the read, as PARS relies on the first base to recover a valid enzyme cleavage point. Reads that were not uniquely mapped were discarded and all genomic locations to which those reads mapped were marked as 'unmappable' due to ambiguity.
Processed data file linked as supplementary file to Series GSE22393 record.

Submission date

Jun 22, 2010

Last update date

May 15, 2019

Contact name

Michael Kertesz

Organization name

Stanford University

Street address

318 Campus Dr.

City

Stanford