GSM2125079: AMB + LF rep 2; Saccharomyces cerevisiae S288C; RNA-Seq
Equal volumes of RNAse zap treated 0.4 μm beads and lyophilised cells were bead beaten in a Minilys tissue homogenizer at 5,000 rpm for 2 min. Cells were cooled on ice at 1 min intervals. Total RNA was then isolated with the Qiagen total RNA mini isolation kit. RNA quality was analysed with a Nanodrop (Thermo Scientific) to ensure an OD260/230 and OD260/280 ratio between 1.8 – 2, and a RNA Quality Indicator (RQI) of at least 8 with a Bioanalyser (Bio-Rad). The concentration was checked using the Qubit BR RNA assay (InVitrogen). For the AMB only treatment, the AMB+LF treatment and their corresponding matching controls, the TruSeq RNA Library prep kit v2 (Illumina) with a poly-A pulldown was used according to the manufacturer's instructions, with 2 μg of total RNA for each of the three biological triplicate samples as input. The libraries were enriched using 15 cycles of PCR and the insert size ranged from 80 to 330 base pair (bp). The libraries were randomized and sequenced in two separate lanes using HiSeq 2000 with the TruSeq v3 SBS reagents to generate 100 bp paired-end reads. The data was demultiplexed using Casava 1.8.2.     For the LF only treatment and the corresponding matching controls, the TruSeq Stranded mRNA-seq prep kit was used to prepare the RNA samples as per protocol using 1ug of total RNA for each of the three biological triplicate samples as input. The libraries were enriched using 12 cycles of PCR to minimize the number of PCR duplicates. The libraries were multiplexed and sequenced on the same flow cell using NextSeq500 v2 reagents to generate 75 bp single reads. The data was demultiplexed using Casava 1.8.2.
GEO Accession
GSM2125079