organism: Schizosaccharomyces pombe
description: Protocols: Cells were grown in EMM medium at 32 degrees C and harvested from liquid cultures before or after removal of nitrogen from the media . 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.). 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: SAMEA2171062
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: 168 hour
attributes.tag: Submitter Id
attributes.value: E-MTAB-1824:MN7_7d
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: fission yeast
attributes.tag: genotype
attributes.value: 972 h-
attributes.tag: sample name
attributes.value: E-MTAB-1824:MN7_7d
attributes.tag: scientific_name
attributes.value: Schizosaccharomyces pombe
attributes.tag: sex
attributes.value: mating type h minus
attributes.tag: strain
attributes.value: JB22