GSM1532599: by_YPD_2; Saccharomyces cerevisiae; RNA-Seq
Total RNA was isolated by a standard hot phenol method and treated with RNase-free DNaseI using Turbo DNA-free kit (Ambion) 10 µg of DNA-free total RNA was desphosphorylated by treatment with Calf Intestinal Alkaline Phosphatase, phenol purified and incubated with Tobacco Acid Pyrophosphatase. Sample was phenol purified and ligated overnight using T4 RNA ligase 1 (NEB) to a DNA/RNA oligo containing a random 8N molecular barcode (CTTTCCCTACACGACGCTCTTCCGATrCrUrNrNrNrNrNrNrNrN). Ligated total RNA was poly(A) purified, and reverse transcribed using random hexamers. Second strand was generated by 1 cycle PCR using oligo Biotin-TATAGCGGCCGCAATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT. Double stranded cDNA was bound to Streptavidin beads, washed, end repaired, dA added and ligated to barcoded P7 linkers as described for the 3’T-fill protocol (PMID: 23295673). The PCR product was size-selected using double Ampure XP size and libraries with an average size of 308bp were submitted for Illumina 50bp paired-end sequencing.
GEO Accession
GSM1532599