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Sample GSM1216865

Query DataSets for GSM1216865

Status

Public on Mar 18, 2014

Title

Wild-type_2

Sample type

SRA

Source name

S. pombe cells, wt

Organism

Schizosaccharomyces pombe

Characteristics

strain: wild-type (ED668)
genotype: h+ ade6-M216 ura4-D18 leu1-32

Growth protocol

Two biological replicates of wild-type (ED668) and dbr1.Δ cultures were grown in YES (yeast extracts plus supplements) media at 32°C till they reached OD600 0.4.

Extracted molecule

total RNA

Extraction protocol

Cells were harvested and total RNA was isolated by hot-phenol extraction, and RNA quality was assessed on a Bioanalyzer instrument (Agilent). Total RNA was treated with DNase (Turbo DNA-free by ambion), and thereafter 4 μg was treated with a beta version of Ribo-Zero™ Magnetic Gold Kit (Yeast) to deplete rRNAs.
RNA-seq libraries were prepared from rRNA-free RNA using a strand-specific library preparation protocol based on an early version of the Illumina TruSeq Small RNA Sample Prep Kit. In brief, rRNA-depleted RNA was fragmented to an average size of ~200nt. Fragmented RNA was 3’-de-phosphorylated with Antartic phosphatase and 5’-phosphorylated with polynucleotide kinase; this treatment prepares RNA fragments for subsequent ligation of Illumina RNA adaptors to their 5’ and 3’ ends using a 3’-RNA ligase and a T4 RNA ligase, respectively. First-strand cDNA was produced using a primer specific for the Illumina 3’-adaptor. The library was amplified with 15 PCR cycles using primers specific for the Illumina adaptors and purified using SPRI-beads (Agencourt, Beckman Coulter). Library size distributions and concentrations were determined on a Bioanalyzer (Agilent). RNA-seq libraries were sequenced on an Illumina HiSeq 2000 instrument at the Core Facility of the Huntsman Cancer Institute (University of Utah).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

wt_2 (Name in ExpressionTable.txt)
total RNA (rRNA depleted)
Strand specific RNA-Seq

Data processing

Sequence reads of 49 base length originating from each sample were aligned, using Bowtie 0.12.725, to the S. pombe genome sequence (Ensembl S. pombe, Build EF1, version 13) and to the corresponding exon-exon junctions database. Up to 3 base-pair mismatches were allowed. Reads that matched multiple loci were removed from further analysis, and the resultant alignment files were processed to generate ‘pile-ups’ against each chromosome. Unmapped reads were used for lariat mapping,.
Searches were performed against the genome sequence combined with a dataset of known exon-exon junctions as defined by Ensembl S. pombe, release-13. To ensure that a 49-base read mapped to a splice junction, only the last 43 bases of the first exon and the first 43 bases of the second exon were considered (if the exon exceeded length 43). In this way, reads that overlapped a junction by less than 6 nucleotides were excluded. Reads that matched to more than one junction or elsewhere in the genome were also discarded.
Genome_build: Ensembl S. pombe, Build EF1, version 13
Supplementary_files_format_and_content: Expression table and RPKM summary for all samples in tab-delimited file ExpressionTable.txt (linked as supplementary file on Series record)

Submission date

Aug 27, 2013

Last update date

May 15, 2019

Contact name

Danny Asher Bitton

E-mail(s)

d.bitton@ucl.ac.uk

Phone

+44(0)203-108-1604

Organization name

University College London