GSM1903957: by_neg2; Saccharomyces cerevisiae; RNA-Seq
All  samples were collected by centrifugation and frozen in liquid N2. Total RNA was isolated by a standard hot phenol method and treated with RNase-free DNaseI using Turbo DNA-free kit (Ambion) Total RNA was isolated by a standard hot phenol method and treated with RNase-free DNaseI using Turbo DNA-free kit (Ambion). 60 µg of DNA-free total RNA was desphosphorylated by treatment with Calf Intestinal Alkaline Phosphatase, phenol purified and splited in 4 aliquots. Samples were treated with 5 units of Tobacco Acid Pyrophosphatase (Epicentre), Cap-Clip Acid Pyrophosphatase (CellScript, cat. no. C-CC15011H), Decapping Pyrophosphohydrolase  (Tebu-bio, cat. no. 436004-35UL) or omitting any decapping treatment.  After that each sample 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 P7. The PCR product were barcoded for multiplexing, size-selected using double Ampure XP size and libraries with an average size of 400-500bp .were submitted for Illumina 50bp single-end sequencing.
GEO Accession
GSM1903957