NextSeq 550 sequencing; RNA-seq of OPI1 knockout yeast cells and the effect of methyl methanesulfonate (MMS) treatment relative to wild-type yeast cells with or without MMS treatment
RNA-seq of OPI1 knockout yeast cells and the effect of methyl methanesulfonate (MMS) treatment relative to wild-type yeast cells with or without MMS treatment
250 mL of cells grown in synthetic complete at an OD600 of 0.4 were vacuum filtered in 0.22 μm nitrocellulose membranes and immediately flash frozen in liquid nitrogen. 1 mL of 1X RIPA buffer was combined into each frozen pellet and lysed by mechanical grinding with a Spex 6750 Freezer Mill at a rate of 10 Hz for 2 minutes. The lysate was then thawed on ice and centrifuged at 2,000 g for 2 minutes at 4oC to remove debris. Cells were grown overnight in synthetic complete media and diluted next day to an OD600 of 0.1 in 250 mL of synthetic complete media in biological duplicates. Cells were grown for at least two cell cycle divisions (to an approximate OD600 of 0.4). Methyl methanesulfonate (MMS) at a final concentration of 0.1% was added to the cells (when indicated) for 1 hour. Reads were demultiplexed using bcl2fastq (version 2.20) and quality of reads was assessed using FastQC (version 0.11.8). 250 μL of cleared lysate was used for RNA 251 extraction by combining it with 250 μL of 2X AE Buffer (100 mM sodium acetate pH 5.2, 20 mM EDTA), 50 μL of 10% SDS and 500 μL of acid phenol: chloroform pH 5.2. The mixture was incubated at 65oC for 10 minutes under 1400 rpm agitation, then transferred to ice for 5 minutes. The solution was then spun at 15,000 g for 15 minutes at 4oC. The supernatant was then transferred to new microcentrifuge tubes and 550 μL of chloroform was added, followed by centrifugation at 15,000 g for 5 minutes at 4oC. The aqueous phase was then transferred to new microcentrifuge tubes and RNA was precipitated in 0.3M sodium acetate and 400 μL of isopropanol after centrifugation at 15,000 g for 20 minutes at 4oC. RNA pellets were washed with 75% ethanol and centrifuged at 15,000 g for 5 minutes at 4oC. Finally, RNA pellets were resuspended in nuclease-free water and submitted to Bioanalyzer High Sensitivity DNA Assay (Agilent) for quality check and quantified using Qubit RNA High Sensitivity (Invitrogen). 1 μg of total RNA was used as input for preparing the libraries for RNA sequencing with Ultra RNA Library Prep II (New England Biolabs), following the recommended workflow. Final cDNA libraries after PCR were quantified by Qubit dsDNA High Sensitivity (Invitrogen).