organism: Schizosaccharomyces pombe
description: Protocols: Fission yeast pat1-114 cells (ade6-M210/ade-M216 pat1-114/pat1-114 h+/h+) were grown to mid-log phase at 25 degree C in Edinburgh Minimal Medium (EMM). Cells were washed twice in EMM without nitrogen source (EMM-N) and cultured for 12h in EMM-N at 25 degree C. The culture was then supplemented with 0.5g/L NH4Cl and shifted to 34 degree C. Samples were collected for RNA preparation just before, and every hour for 8 hours after shifting the culture to 34 degree C. Equal RNA amounts from each time-point were pooled and used for sequencing library preparation. Total RNA was isolated from S. pombe cells using a hot phenol method followed by phenol-chloroform extractions, and precipitation as described (Lyne et al., 2003, BMC Genomics 4, 27.). Equal amounts of RNA from each time point were pooled together. 10ug total RNA was used as starting material for strand-specific RNA-seq library preparation using an unreleased early version provided by Illumina of the Illumina TruSeq Small RNA Sample Prep Kit. Briefly: poly(A)+ RNA was enriched by two rounds of poly(dT) Sera-Mag magnetic beads purification. Enriched poly(A)+ RNA was then 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 finally amplified with 15 cycles of PCR using primers specific for the Illumina adaptors and purified using SPRI-beads (Agencourt, Beckman Coulter). Libraries size distributions and concentrations were determined on an Agilent Bioanalyzer
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2013-09-01T17:01:55Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-03-08T16:41:23Z
attributes.tag: External Id
attributes.value: SAMEA2171063
attributes.tag: INSDC center name
attributes.value: Department of Genetics, Evolution, and Environment & Cancer Institute, University College London
attributes.tag: INSDC first public
attributes.value: 2013-09-01T17:01:55Z
attributes.tag: INSDC last update
attributes.value: 2018-03-08T16:41:23Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: PAT cells culture time
attributes.value: 1 hour
attributes.tag: PAT cells culture time
attributes.value: 7 hour
attributes.tag: PAT cells culture time
attributes.value: 2 hour
attributes.tag: PAT cells culture time
attributes.value: 6 hour
attributes.tag: PAT cells culture time
attributes.value: 0 hour
attributes.tag: PAT cells culture time
attributes.value: 3 hour
attributes.tag: PAT cells culture time
attributes.value: 5 hour
attributes.tag: PAT cells culture time
attributes.value: 4 hour
attributes.tag: PAT cells culture time
attributes.value: 8 hour
attributes.tag: Submitter Id
attributes.value: E-MTAB-1824:pooled_PAT_cells
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: fission yeast
attributes.tag: genotype
attributes.value: ade6-M210/ade-M216 pat1-114/pat1-114 h+/h+
attributes.tag: sample name
attributes.value: E-MTAB-1824:pooled_PAT_cells
attributes.tag: scientific_name
attributes.value: Schizosaccharomyces pombe
attributes.tag: sex
attributes.value: mating type h plus
attributes.tag: strain
attributes.value: pat1-114