Illumina HiSeq 2000 sequencing; RNA-Seq of wild type yeast and several knockout strains in different chemically defined media
RNA-Seq of wild type yeast and several knockout strains in different chemically defined media
Yeast cultures were harvested at OD600 ~1 by mixing 1:1 with cold methanol (ethanol and dry ice bath), centrifuged at -9°C, and pellets were stored at -80°C. Total RNA was extracted using RNeasy Mini Kit (QIAGEN). Customized steps included homogenization with acid washed glass beads using FastPrep-24 bead beater (4 m/s setting, for 1 min) and on column DNA digestion step using Turbo DNAse (Ambion). RNA quality was verified using 1% agarose gel visualization and Agilent 2100 bioanalyzer, quantified with Qubit 2.0 fluorometer (Invitrogen) using the Qubit RNA BR assay kit.  cDNA library was prepared from starting amount of 500 ng total RNA according to Illumina's RNA Seq protocol using Beckman Biomek FX laboratory automation workstation.