GSM1406441: Sp early stationary phase (ES) RNA-Seq; Saccharomyces paradoxus; RNA-Seq
Total RNA was isolated with Yeast RNA Purification Kit (MasterPure™ Epicentre). Genomic DNA contamination was removed with Turbo DNase (Ambion) followed by phenol chloroform extraction. The cDNA libraries for RNA samples were constructed using TruSeq RNA Sample Preparation Kits v2 based on the guide (Illumina, Cat. No RS-122-2001). PolyA-containing mRNA was purified using 50 μl RNA Purification Beads for each sample. The strand-specific cDNA library was constructed by dUTP second-strand method (Parkhomchuk et. al., 2009). The purified RNA fragments were reverse-transcribed into first-strand cDNA using SuperScript II Reverse Transcriptase (Invitrogen, Cat. No 18064-014) and random primers (Illumina, Cat. No. RS-122-2001). The first strand cDNA/RNA hybrid was precipitated with ethanol and ammonium acetate. The second-strand cDNA synthesis was performed in a 80 μl reaction volume using NEBNext® mRNA Second Strand Synthesis Module (NEB, NEB # E611lL) and dNTP/dUTP mix (Fermentas , Cat. No. R0251, dTTP→dUTP ) at 16°C for 2.5 hours. The DNA fragments were purified by QIAquick PCR Purification Kit (Qiagen, Cat. No 28106) and treated with T4 DNA polymerase, E. coli DNA polymerase I Klenow fragment and T4 Polynucleotide Kinase in a 30 μl reaction using the buffer and the enzyme in the kit (Illumina, Cat. No RS-122-2001) at 37°C for 30 min. The blunt phosphorylated DNA fragments were purified by the MinElute PCR Purification Kit (Qiagen, Cat. No 28006) and treated with Klenow fragment of DNA polymerase lacking exonuclease activity in the presence of dATP to add an adenine overhang to the 3’ ends of each strand in a 30 μl reaction using the kit (Illumina, Cat. No. RS-122-2001) at 37°C for 30 min. Adapters required for sequencing on the Illumina platform were added to DNA fragments by ligation in a 37.5 μl reaction using the kit (Illumina, Cat. No. RS-122-2001). The ligation products were purified by the MinElute PCR Purification Kit (Qiagen, Cat. No 28604) and eluted the ligation products in the size range from 350 to 450 bp from the gel. These products were precipitated and resuspended in 21 μl TrueSeq Resuspension Buffer using QIAquick Gel extraction Kit (Qiagen, Cat. No 28704) and treated with USER™ Enzyme mixture (Uracil-Specific Excision Reagent, NEB, Cat. No M5505L) to degrade the dUTP in the coding strand (second strand). The adapter-modified DNA fragments were amplified by the kit (Illumina, Cat. No. RS-122-2001). The amplified products were purified by Agencourt AMPure XP (Beckman, Cat. No A63881) and collected in 30 μl Resuspension Buffer (Qiagen, Cat. No 28604). After quantification by Quant-iT dsDNA HS Assay Kit (Invitrogen, Cat. No Q32851) and KAPA QPCR Library Quantification Kit (KAPABiosystem. Cat. No kk4822), calculation of molar concentration and quality examination by Expersion DNA 1K Analysis Kit (Bio-Rad Cat. No 700-7107), this DNA library was prepared for sequencing.