GSM2973966: trm44_RepA_OD12; Saccharomyces cerevisiae; OTHER
Total yeast RNA was isolated by hot acid phenol extraction followed by isopropanol precipitation. PolyA+ mRNA was isolated from 10-15mg of yeast total RNA by two rounds of selection on oligo-dT cellulose beads according to the manufacturer's instructions (NEB). RNA fragmentation was performed in 10mM ZnCl2 at 95C for 55s. RNA fragments were precipitated, dephosphorylated with CIP and PNK, and separated by denaturing (8% urea-TBE) polyacrylamide gel electrophoresis. Gel-purified RNA fragments (60-70nt, 70-80nt, 80-90nt) were eluted overnight with rocking in RNA elution buffer (300mM NAOAc pH 5.5, 1mM EDTA, 100 U/ml RNasin (Promega) and ligated to a pre-adenylated adaptor using T4 RNA ligase (NEB) at 22°C for 2.5h, followed by precipitation. Reverse transcription was completed using AMV-RT (Promega) and the reverse transcription primer using the following conditions. RNA and RT primer were denatured and annealed in reverse transcription buffer (50 mM Tris-Cl pH 8.6, 60mM NaCl, 10mM DTT). After annealing, the reverse transcription reaction was split in equal halves for the restrictive and permissive conditions. The restrictive reaction received a final concentration of 2.5mM dNTPs, 6mM MgCl2 and 7.1% DMSO, whereas the permissive reaction received a final concentration of 4mM dNTPs, 20mM MgCl2 and 7.1% DMSO. Reactions were carried out at 42°C for 1h, prior to degradation of RNA with the addition of NaOH at 85°C. Truncated cDNAs were size-selected and purified on an 8% urea-TBE PAGE gel, followed by elution from gel slices in DNA elution buffer (300mM NaCl, 10mM Tris-HCl, pH 8.0) overnight at room temperature. cDNAs were circularized with circLigase (Epicentre) and amplified by PCR (Phusion; NEB) with the forward primer  and a barcoded reverse primer . PCR products were gel-purified, precipitated and sequenced on an Illumina HiSeq 2000.
GEO
GSM2973966