NextSeq 550 paired end sequencing; Microfluidic platform for monitoring Saccharomyces cerevisiae mutation accumulation assay
Microfluidic platform for monitoring Saccharomyces cerevisiae mutation accumulation assay
MA lines obtained by classical plating and microfluidic approaches were conserved in a frozen stock and streaked on YPD medium for 48 hours at 30°C. In addition, the parental strains were also included in genome sequencing analysis. Colonies were then inoculated from the solid medium onto 10mL of liquid YPD overnight on a 30°C 180 rpm shaking incubator until saturation. Cells were then pelleted and resuspended in 200 μL of a buffer containing 10mM Tris-HCl pH 8.0, 1mM EDTA, 100 mM NaCl, 1% SDS, 2% Triton X100, for DNA extraction with glass beads (Sigma Aldrich) and Phenol:Chloroform:Isoamyl Alcohol (25:24:1) (Sigma-Aldrich)  and vortexed at room temperature for 10 minutes. 200μL of Tris-EDTA (Sigma-Aldrich) buffer was added and this was centrifuged at 1300rpm for 5 minutes at room temperature. The supernatant was then precipitated with twice the volume of cold absolute Ethanol (VWR Chemicals) and 0.1 volume of 3M NaOAc (Sigma Aldrich). The solution was precipitated for 20 minutes at -80°C, centrifuged at 4°C and the pellet was washed with cold Ethanol 70% (VWR Chemicals) and then resuspended in water. The solution was treated with RNAse at 50μg/mL (Thermofisher Scientific) at 37°C for 1hour. The solution was precipitated with Ethanol for 20 minutes at -80°C, centrifuged at 4°C and the pellet was washed in cold Ethanol 70% (VWR Chemicals) and then resuspended in water. DNA was quantified using Nanodrop (Thermoscientific) and genomic DNA samples from 4 parallel MA lines for each strain were merged in equal quantities for library preparation. DNA was then sonicated with Covaris sonicator using the program of a Duty Factor of 10%, Peak incident power 175W, Cycles per burst 200 and a time of 180 seconds. Library preparation for genomic DNA was performed on IP-Star system using MicroPlex Library Preparation Kit v2 (Diagenode), following manufacturer's instructions.