RNAseq:S.cerevisiae BY4741
BY4741 and yjl175w_ko were grown in YPD at 30 and sampled in logarithmic growth phase. The cells were collected by centrifugation at 10,000 g for 10 mins. Total RNA from 0.5 g of collected yeast cell samples was extracted by using a FastRNA Pro Soil-Direct Kit (Qbiogene, Solon, OH) according to the manufacturers instructions. A DNase-treatment was conducted to remove genomic DNA from total RNA by using Recombinant DNaseI (RNase-free) (TaKaRa, Kyoto, Japan). After ethanol precipitation, the purified total RNA was stored at 80 until the subsequent step. RNA purity and concentrations were estimated by a NanoDrop spectrophotometer (Thermo Scientific, Yokohama, Japan). RNA quality and quantity were estimated by a Bioanalyzer (Agilent, Tokyo, Japan). cDNA from the purified mRNA was synthesized using a cDNA Synthesis Kit (TaKaRa). First, the poly(A) RNA was reverse-transcribed with an Oligo(dT)-T7 primer containing a T7 promoter sequence, and then the double-stranded cDNA was synthesized. Next, the cDNA templates were transcribed in vitro with T7 RNA polymerase (TaKaRa), yielding large amounts of antisense RNA (aRNA). Finally, aRNA was further reverse- transcribed to cDNA with biotinated oligo (dT) primer for Next-generation sequencing.