GSM6165894: upf1_CHX_T_2; Saccharomyces cerevisiae; RNA-Seq
Total RNA was extracted using standard hot phenol procedure. Extracted RNA was ethanol-precipitated, resuspended in nuclease-free H2O (Ambion) and quantified using a NanoDrop 2000c spectrophotometer and/or a Qubit fluorometer with the Qubit RNA HS Assay kit (Life Technologies). For each sample, 1.5 µg of total RNA was mixed with 3 µl of diluted ERCC RNA spike-in mix and then digested for 1h at 30°C with 1 unit of Terminator 5´-Phosphate-Dependent Exonuclease (Epicentre) in 1X Reaction Buffer A containing 10 units of SUPERase-In RNase inhibitor (Invitrogen). After phenol/chloroform extraction, Terminator-digested RNA was precipitated with ethanol, and then resuspended in nuclease-free H2O. Libraries were prepared using the TruSeq Stranded mRNA Sample Preparation Kit (Illumina) and the IDT for Illumina – TruSeq RNA UD indexes (Illumina).