GSM4826141: Spt4-FRB_DMSO_RNA-seq; Saccharomyces cerevisiae; RNA-Seq
Cells were filtered and flash frozen in liquid nitrogen. 4 parts of the frozen S.cerevisiae pellet were spiked-in with one part of frozen S.pombe (containing a FLAG-tagged Rpb9) pellet (4:1 by weight). The combined pellet was ground with mixer mill (6 cycles, 3 min, 15 hz). The grindate was lysed in 20 mM HEPES (pH 7.4), 110 mM KOAc, 0.5 % Triton-X-100, 0.1 % Tween 20, 10 mM MnCl2, 1x proteinase inhibitors (Roche; complete, EDTA-free), 50 U/ml SUPERase.In RNase inhibitors (Invitrogen), 132 U/ml DNase I (Promega) in ice for 20 min, and cleared from cell debris (16,000 g, 10 min). Total RNA was extracted from supernatant using Qiagen miRNA kit according to the manufacturer's instructions. Large rRNA was depleted using Ribominus transcriptome isolation kit for yeast (Invitrogen) and standard NEBNext Ultra II Directional RNA Library Prep Kit for Illumina.