RNA-Seq profiling of S. Cerevisiae WT BY4741 cells and MOH1-knockout mutants of BY4741 cells (moh1∆) after growth on YPD-Agar for 40 hours
RNA-Seq profiling of S. Cerevisiae WT BY4741 cells and MOH1-knockout mutants of BY4741 cells (moh1∆) after growth on YPD-Agar for 40 hours
S.cerevisiae BY4741 (MATa, his3∆1, leu2∆0, met15∆0, ura3∆0) strain as the wild-type strain (WT) and BY4741 MOH1 knockout mutant strain (moh1∆; MATa, his3∆1, leu2∆0, met15∆0, ura3∆0, Moh1Δ::KanMX4) were used for the RNA-Sequencing. Cells grown overnight in YPD at 30 °C with reciprocal shaking at 180 rpm were sub-cultured into fresh YPD at a 1:100 dilution and grown until OD600 reached 0.4-0.6. Cells were subsequently spotted at serial dilution on YPD-agar plates. Plates were incubated at 30 °C for 40 hours. Cells were collected with scraper.  Collected cells were pelleted with centrifugation.  To dissolve cell pellets, 25 μl of 20% SDS and 400 μLof ice-cold Acetate-EDTA (AE) buffer containing 50 mM sodium acetate and 10 mM EDTA (pH=8) were added to the cell pellets. Subsequently, 500 μl of acidic 25:24:1 phenol:chloroform:isoamyl alcohol (PCI) solution was added to the cell suspension. The cell suspension was incubated at 65°C for 15 minutes, followed by incubation on ice for 10 minutes. For phase separation, samples were centrifuged at 14000 rpm at 4°C for 15 minutes, and the liquid phase was transferred to a sterile tube containing 500 μLof PCI. The mixture was vortexed for 20 seconds and incubated on ice for 10 minutes. Following centrifugation of the samples at 14000 rpm at 4 °C for 15 minutes, the liquid phase was transferred into a new sterile tube. Then, 1/10 volume of sodium acetate (pH 5.3) and three volumes of 100% ethanol were added. Samples were then incubated at -80 °C for 30 minutes. RNA was precipitated by centrifugation at 14000 rpm at 4 °C for 15 minutes and dissolved in diethylpyrocarbonate (DEPC)-treated water. RNA samples were also treated with DNase I to degrade any remaining DNA. The purity and concentration of RNA were determined using a Nanodrop 2000 spectrophotometer (Thermo-Fisher Sci., California, USA) with A260/A280 and A260/A230 ratios.  MGIEasy Fast RNA Library Prep Set was used. The first step in the workflow involves purifying the poly-A containing mRNA molecules using poly-T oligo-attached magnetic beads. Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. This is followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then have the addition of a single 'A' base and subsequent ligation of the adapter. The products are then purified and enriched with PCR amplification. We then quantified the PCR yield by Qubit and pooled samples together to make a single strand DNA circle (ssDNA circle), which gave the final library.