GSM1892908: WT_3; Saccharomyces cerevisiae; RNA-Seq
Total RNA was isolated using RiboPure™-Yeast Kit (Life Technologies). To eliminate residual DNA, total RNA was treated with 2 μl DNase I (TURBO DNA-free™ Kit; Life Technologies) for 45 min at 37°C. RNA was digested once more using DNase I (RNase-Free DNase set; Qiagen) for 10 min at room temperature before purification using the RNeasy® Kit (Qiagen). Prior to library construction for RNA-seq, the quality of RNA was ascertained using Bioanalyzer (Agilent), and the RNA integrity number (RIN) for the samples ranged from 6.8−8.1. Library construction is performed using the Illumina TruSeq Stranded Total RNA Sample Preparation Kit with Ribo-Zero™ Magnetic Gold Kit (Yeast)  (cat# RS-122-2401, RS-122-2402) as described below.  Briefly, Ribosomal RNA is removed from total RNA samples (100 ng to 4 ug) using biotinylated Ribo-Zero oligos attached to magnetic beads that are complimentary to yeast cytoplasmic  and chloroplast rRNA. Following purification, the rRNA-depleted sample is fragmented with divalent cations under elevated temperatures and primed with random hexamers in preparation for cDNA synthesis.  First strand reverse transcription is accomplished using Superscript II Reverse Transcriptase (Invitrogen cat#18064-014).  Second strand cDNA synthesis is accomplished using DNA polymerase I and Rnase H under conditions in which dUTP is substituted for dTTP, yielding blunt-ended cDNA fragments in which the second strand with dUTP.  An A-base is added to the blunt ends as a means to prepare the cDNA fragments for adapter ligation and block concatamer formation during the ligation step.  Adapters containing a T-base overhang  are ligated to the A-tailed DNA fragments.  Ligated fragments are PCR-amplified (12-15 cycles) under conditions in which the pcr reaction enables amplification of the first strand cDNA product, whereas attempted amplifciation of the second strand product stalls at dUTP bases and therefore is not represented in the amplified library.  The PCR-amplified library is purified using Agencourt AMPure XP beads (Beckman Coulter Genomics cat#A63881).  Following amplification, the library is purified by bead based methodologies.  The concentration of the amplified library is measured with a NanoDrop spectrophotometer and an aliquot of the library is resolved on an Agilent 2200 Tape Station using a D1K (cat# 5067-5361 and 5067-5362) or a High Sensitivity D1K (cat# 5067-5363 and 5067-5364) assay to define the size distribution of the sequencing library.  Libraries are adjusted to a concentration of approximately 10 nM and quantitative PCR is performed using the KapaBiosystems Kapa Library Quant Kit (cat# KK4824) to calculate the molarity of adapter ligated library molecules.  The concentration is further adjusted following qPCR to prepare the library for Illumina sequence analysis. The library was then subjected to 50bp single read sequencing in an Illumina Hiseq2000 sequencer.