title: GSM1466925: T4V replicate 2; Saccharomyces cerevisiae; RNA-Seq
library_construction_protocol: For RNA sequencing analyses, the integrity of total RNA was measured by Agilent Bioanalyzer. RNA samples with an integrity number >8.0 were used for further processing. Total RNA was subjected to 2 rounds of poly(A) selection using oligo-dT magnetic beads (NEB) followed by fragmentation using Ambion’s RNA fragmentation kit at 70°C for 5 min. Fragmented RNA was purified by ethanol precipitation followed by dephosphorylation using recombinant shrimp alkaline phosphatase (NEB) and then phosphorylation using T4 kinase (NEB). Phosphorylated RNA was then purified by RNeasy kit (Qiagen) and was sequentially ligated to a 5′-adenylated 3′ adapter (5′-/5rApp/TGGAATTCTCGGGTGCCAAGG/3ddC/) with the truncated T4 RNA ligase 2 (NEB) and to a 5′ adapter (5′- GUUCAGAGUUCUACAGUCCGACGAUC) with T4 RNA ligase 1 (NEB). The resultant RNA was reverse-transcribed to cDNA with Superscript III (Invitrogen), and the cDNA was amplified by 12 cycles of PCR with Phusion high fidelity polymerase (NEB). Amplified cDNAs were separated on 8% polyacrylamide gels. cDNAs containing an inset size ~100 nt were purified from the gel based on the size. The quality and quantity of the cDNA library were evaluated by Agilent Bioanalyzer and qPCR analysis. The strand-specific cDNA libraries generated by this protocol were sequenced on an Illumina Genome Analyzer IIx.