GSM1604420: Rph1- Old (S3O, Exp 9); Saccharomyces cerevisiae; RNA-Seq
mRNA was purified from 5ug total RNA using the Dynabeads mRNA Direct kit (Ambion). Since yeast genes are relatively short (average gene size ~1.3Kb), we chose to fragment the mRNA to ~40-50bp rather than the conventional 200-300bp size using RNA fragmentation reagents from Ambion. We believe that this gives better resolution over gene bodies to quantify 5’ and 3’ reads for estimation of cryptic transcripts. To do this, the fragmented RNA was phosphorylated by T4 PNK (NEB) and then size selected on a 15% polyacrylamide gel containing urea. Following size selection, the entire RNA was used as input in the TruSeq small RNA sample preparation protocol (Illumina). Final size selection of amplified library was done using 6% polyacrylamide gels.
GEO Accession
GSM1604420