Schizosaccharomyces pombe
Protocols: Cells were grown in EMM (Moreno et al., 1991, Methods Enzymol. 194, 795-823) at 32C, 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, and polyadenylated RNA was ourified using oligo(dT) magnetic beads (Life Technologies) following the manufacturer's protocol. Total RNA was fragmented by mixing 20 ul of RNA 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. 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.
ENA-FIRST-PUBLIC
2014-12-01T17:03:35Z
ENA-LAST-UPDATE
2018-03-08T19:12:40Z
External Id
SAMEA2624972
INSDC center name
Department of Biochemistry, University of Cambridge, UK
INSDC first public
2014-12-01T17:03:35Z
INSDC last update
2018-03-08T19:12:40Z
INSDC status
public
Submitter Id
E-MTAB-2695:RBP26
broker name
ArrayExpress
common name
fission yeast
genotype
SPAC30D11.14delta
sample name
E-MTAB-2695:RBP26
scientific_name
Schizosaccharomyces pombe