GSM1464191: Lib75PUpus7rep1_input; Saccharomyces cerevisiae; RNA-Seq
Enrichment of polyadenylated RNA (polyA+ RNA) from total RNA was performed using one round of enrichment using Oligo(dT) dynabeads (Invitrogen) according to the manufacturer’s protocol. CMC-treatment was performed essentially as described in (Bakin and Ofengand, 1993; Bakin and Ofengand, 1998). Specifically, pelleted polyA+ RNA was resuspended in 30 mL of 0.17M CMC in BEU buffer (50mM bicine, pH 8.3, 4 mM EDTA, and 7M urea) or in 30 mL of BEU buffer as input at 37°C for 20 min. Reaction was stopped with 100 mL of 0.3M NaOAc and 0.1 mM EDTA, pH 5.6 (Buffer A), 700 mL of EtOH, and 1 mL of glycogen. After chilling for 5 min in dry ice, the pellet was recovered, washed with 70% EtOH, dissolved in 100 mL of Buffer A, and reprecipitated with 300 mL of EtOH and 1 mL of glycogen. After washing as above, the pellet was dried, dissolved in 40 mL of 50 mM sodium bicarbonate, pH 10.4, and incubated at 37°C for 3 hours. The RNA was precipitated by addition of 100 mL of Buffer A, 700 mL EtOH, and 1 mL of glycogen. The pellet was washed with 70 % EtOH and then dissolved with water for subsequent library preparation. The mRNA was chemically fragmented into ~80-150 nt-long fragments by performing a short fragmentation (30 seconds) using RNA fragmentation reagent and stop solution (Ambion). RNA was subjected to FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific), followed by a 3’ ligation of an RNA adapter using T4 ligase (New England Biolabs). Ligated RNA was reverse transcribed using AffinityScript Multiple Temperature Reverse Transcriptase (Agilent) or SuperScript III (Life Technologies), and the cDNA was subjected to a 3’ ligation with a second adapter using T4 ligase. The single-stranded cDNA product was then amplified for 9-12 cycles in a PCR reaction. Libraries were sequenced on Illumina HiSeq 2500 platforms generating paired end reads (30 bp from each end).