organism: Schizosaccharomyces pombe
description: Protocols: Cells were grown in yeast extract medium (Moreno et al., 1991, Methods Enzymol. 194, 795-823) at 32C, incubated for 5 minutes with 100 ug/ml cycloheximide, pelleted at 4C and frozen in liquid N2 RNA was extracted using a hot phenol extraction method as described in Lyne et al. (2003) BMC Genomics 4:27. Polyadenylated RNA was purified from 150 ug of total RNA using oligo-dT25 magnetic beads (Life Technologies) following the manufacturers instructions. Purified mRNA was fragmented by mixing 20 ul of mRNA with 20 ul of 2X alkaline fragmentation buffer (2 mM EDTA, 100 mM Na2CO3) followed by incubation for 15 minutes at 95C. Samples were run on 15% TBE-urea gels (Life Technologies), and fragments of around 28 nucleotides were extracted from the gel. Both mRNA and ribosomal footprint gel-purified samples were processed identically. RNA samples were purified using Purelink RNA microcolumns (Life Technologies) as described by the manufacturer, except that the samples were initially passed through the column in the presence of 70% ethanol (to favour binding of small RNAs). Samples were then treated with poly nucleotide kinase (PNK, Fermentas) and polyadenylated using 12 units of poly-(A) polymerase (NEB) at 37C for 45 minutes. Reverse transcription reactions were performed using custom primers containing an anchored oligo(dT), 4 nucleotides of known sequence used for multiplexing, and 5 random nucleotides that serve as unique molecular identifiers. Reverse transcription products were gel-purified and circularised using CircLigase II (Epicentre), and amplified by PCR with custom library primers for 12 or 15 cycles.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2014-06-03T17:04:08Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-03-08T17:02:57Z
attributes.tag: External Id
attributes.value: SAMEA2292866
attributes.tag: INSDC center name
attributes.value: Department of Biochemistry, University of Cambridge, UK
attributes.tag: INSDC first public
attributes.value: 2014-06-03T17:04:08Z
attributes.tag: INSDC last update
attributes.value: 2018-03-08T17:02:57Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-2176:wt_1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: fission yeast
attributes.tag: sample name
attributes.value: E-MTAB-2176:wt_1
attributes.tag: scientific_name
attributes.value: Schizosaccharomyces pombe
attributes.tag: strain
attributes.value: 968 h90