title: Wild-type at vegetative growth
organism: Schizosaccharomyces pombe
description: Wild-type cultures were grown at 32°C in YES medium
attributes.tag: Data processing step
attributes.value: Sequence reads of 50 or 51 base length originating from each sample were aligned, using Bowtie 0.12.725, to the S. pombe genome sequence (Ensembl S. pombe, Build EF1, release 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. Importantly, throughout our exon-skipping analysis we used only canonical and non-canonical exon-exon junction reads and reads that straddle the exon-intron boundaries, while the remaining reads were ignored. Therefore, expression levels are not provided. Searches were performed against the genome sequence combined with a database of all possible exon-exon junction sequences that could be generated from Ensembl S. pombe annotation (release-13). To ensure that an 'X'-base read is mapped to a splice junction (see Supplementary Table S1 for exact read length), only the last ('X'-6) bases of the first exon and the first ('X'-6) bases of the second exon were considered (if the exon exceeded length 'X'-6). 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.
attributes.tag: ENA first public
attributes.value: 2015-03-24
attributes.tag: ENA last update
attributes.value: 2014-09-25
attributes.tag: External Id
attributes.value: SAMEA2785377
attributes.tag: INSDC center alias
attributes.value: Dbitton
attributes.tag: INSDC center name
attributes.value: Dbitton
attributes.tag: INSDC first public
attributes.value: 2015-03-24T17:04:20Z
attributes.tag: INSDC last update
attributes.value: 2014-09-25T16:43:35Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Library constraction protocol
attributes.value: RNA-seq libraries were prepared 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 (apart from nmt1-rpb1 sequenced on Genome Analyzer II).
attributes.tag: Submitter Id
attributes.value: Veg.growth_sample_127
attributes.tag: common name
attributes.value: fission yeast
attributes.tag: extraction protocol
attributes.value: Cells from all cultures were harvested at mid-log phase (optical density, OD600nm = 0.5) and total RNA was isolated by hot-phenol extraction, and RNA quality was assessed on a Bioanalyzer instrument (Agilent). For ribosomal depleted cultures: total RNA was treated with DNase (Turbo DNA-free by ambion), and thereafter 4 μg was treated with a Ribo-Zero™ Magnetic Gold Kit (Yeast) to deplete rRNAs. For poly-(A) enriched cultures: poly(A)+RNA was enriched by two rounds of poly(dT) Sera-Mag magnetic bead purification.
attributes.tag: genotype
attributes.value: leu1-32 h-
attributes.tag: library strategy
attributes.value: Strand Specific RNA-seq. poly-(A) enriched
attributes.tag: sample name
attributes.value: Veg.growth_sample_127