title: pka1 -deletion-cAMP-dependent protein kinase catalytic subunit mutant at early-mid stationary phase
organism: Schizosaccharomyces pombe
description: Cells were grown in EMM 32°C; Harvested deep in stationary phase when viability dropped to 87% (assesed by CFU)
attributes.tag: Data processing step
attributes.value: Reads were aligned to the fission yeast genome with the exonerate software (Slater and Birney 2005) and reads matching to multiple locations in the genome were assigned at random to only one of these locations. Reads containing up to 5 mismatches (not clustered at read ends) were kept for further analysis (���Genomic set�۝). The remaining sequencing reads were then mapped against fission yeast spliced exons and filtered as above (���Spliced set�۝). Pools of both sets of reads were used for further analyses. Between 20 and 50 million mappable reads were obtained for each library (~80-85% of total reads were mappable).Relative sequencing expression scores were calculated for annotated features (spliced transcripts and introns), using the genome annotation available in PomBase http://www.pombase.org/. The number of reads mapping to each feature was divided by the length of the feature per kilobase and read depth per million mapped reads (reads per kilobase of transcript per million reads mapped, RPKM). RPKMs for annotated features correlated strongly between biological replicates (Pearson's correlation coefficient >0.98).
attributes.tag: ENA first public
attributes.value: 2015-03-24
attributes.tag: ENA last update
attributes.value: 2014-09-29
attributes.tag: External Id
attributes.value: SAMEA2786179
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: 2014-09-29T13:44:44Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: pka1_Glu_87_sample_188
attributes.tag: common name
attributes.value: fission yeast
attributes.tag: extract 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).
attributes.tag: genotype
attributes.value: h- pka1::KanMX4
attributes.tag: growth protocol
attributes.value: All RNA processing mutant cell cultures were harvested at mid-log phase (optical density, OD600nm = 0.5). With the exception of conditional mutants (rrp6-ts, dis3-54), all RNA processing mutant cultures were grown at 32�C in rich medium (YES) For meiotic time course samples: briefly, S. pombe pat1-114 cells were grown to mid-log phase before being shifted to Edinburgh minimal media (EMM) without nitrogen. Cells were incubated at 25�C overnight to synchronise them in G1 phase. Meiosis was induced by addition of NH4Cl to final concentration of 0.5g/L and incubation at 34�C. Cells were harvested and RNA extracted hourly after induction of meiosis. A pool of meiotic RNA was created by pooling equal amounts from all time-points (0, 1, 2, 3, 4, 5, 6, 7, 8h). For glucose starvation experiments, wild-type (WT) cells were grown in EMM at 32�C, 2% glucose. A sample representing 100% survival was harvested when cultures reached a stable maximal density. Measurements of Colony Forming Units (CFUs) were conducted every 24 hours after this initial time point, and another sample harvested when cultures reached 50% survival. pka1 samples under glucose starvation were prepared in the same way, with an additional sample taken when cultures reached 87% survival (this is the same time point at which a WT culture run in parallel reached 50% survival). For nitrogen starvation experiments, cells were grown in EMM at 32�C to a stable maximal density. Cells were then washed twice in EMM without nitrogen source (NH4Cl) and cultured in EMM without nitrogen at 32�C. Cells were harvested at 24 hours and 7 days after nitrogen removal.
attributes.tag: library construction protocol
attributes.value: Strand-specific RNA-seq libraries were prepared from poly(A)-enriched (poly(A)+) RNA using an unreleased early version of the Illumina TruSeq Small RNA Sample Prep Kit. Briefly, 10mg of total RNA was used as starting material and poly(A)+ RNA was enriched by two rounds of poly(dT) Sera-Mag magnetic bead purification. RNA was fragmented to an average size of ~200 nucleotides (nt). Fragmented RNA was 3�۪ de-phosphorylated with Antarctic phosphatase and 5�۪ phosphorylated with polynucleotide kinase; this treatment prepared 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 Bioanalyser (Agilent).
attributes.tag: library strategy
attributes.value: Strand specific RNA-seq. poly(A)-enriched
attributes.tag: sample name
attributes.value: pka1_Glu_87_sample_188