GSM1406457: Cycloheximide replicate 2; Saccharomyces cerevisiae; RNA-Seq
Cells were harvested by filtration then frozen in liquid N2 and pulverized using a Retsch mixer mill. Lysis buffer was added to cell powder and lysates were clarified by centrifugation. RNAse I was added to cell lysate. After RNAse digestion, lysate was sedimented in a linear 10%-50% sucrose gradient at 35000rpm for 3 hours. Gradients were fractionated and the monosome peak fraction was collected. RNA was purified using a miRNeasy Mini kit from Qiagen. RNA was size selected using 18 and 34 nt size markers then treated to remove the 3’ phosphate and ligated to the Universal miRNA cloning linker from New England Biolabs. Ligated fragments were reverse transcribed using a primer complementary to the cloning linker which introduced sequencing adapters. RNA template was removed and cDNA was circularized using CircLigase ssDNA ligase from Epicentre. Ribosomal RNAs were removed by hybridization to subtraction oligos. Libraries were amplified with PCR. Libraries were then sequenced on an Illumina Genome Analyzer 2 or an Illumina HiSeq 2000.