GSM5235676: ras1 Low-glucose rep01 [RAS1_L1]; Saccharomyces cerevisiae; RNA-Seq
500 μL of cell culture was aliquoted into a 1.7 mL microfuge tube and centrifuged at 1000 x g for 1 min at 4 °C. After discarding supernatant, the cell pellet was flash frozen by liquid nitrogen and stored at -80 °C. Cell pellets were resuspended with 600 μL buffer RLT 1% (v/v) 2-mercaptoethanol from the QIAGEN RNeasy Mini Kit (Cat No.: 74106) and then added into 2 mL OMNI prefilled ceramic bead tubes (SKU: 19-632). Tubes were loaded onto an OMNI Bead Mill Homogenizer (SKU:19-040E) for 3 beating cycles. For each cycle, samples were agitated at 5 m/s for 1 min at 4 °C and then cooled on ice for 3 min between each cycle. The resulting lysate was clarified by centrifugation at 11,000 xg and then used for total RNA extraction with QIAGEN RNeasy Mini Kit (Cat No.: 74106) with on-column DNase digestion according to manufacturer's instructions. Extracted total RNA for each sample was checked for purity and quantified with Invitrogen Qubit 2.0 Fluorometer (Cat No.: Q32866) and Qubit RNA HS Assay kit (Cat No.: Q32855) according to manufacturer's instructions. KAPA Stranded mRNA Seq Kit (96 RXN) (Cat#07962207001) was used with 3 ug of total RNA for the construction of sequencing libraries. KAPA standard protocol was followed. Customized unique dual indexed barcodes from Perkin Elmer were used.