RNA-seq of Candida strain P57055-dhh1 in YPD
Overnight cultures (YPD media, 30C) were diluted to OD600 = 0.2 and grown to OD600 = 0.8 in YPD before harvesting 20 mL by centrifugation and freezing in liquid nitrogen. Cell pellets were resuspended in 1 mL ice cold RNA buffer (4M NaCl, 1M Tris-HCl pH 7.5, 1 mM EDTA). 500 uL of Acid-Phenol CHCl3 (pH 4.5) and 250 uL of zirconia beads were added to 500 uL of resuspended cells in 2 mL eppendorf tubes. Cells were disrupted by ten cycles of 30s vortexing and 1 minute on ice. After centrifugation for 2 minutes, the aqueous phase was removed and back-extracted with an equal volume of CHCl3-IAA. The resulting aqueous phase was precipitated with 3 volumes of ethanol at -80C. Pellets were washed with 70% ethanol and resuspended in 100 uL of ultrapure water. 5 ug of total RNA was treated with TurboDNase (Invitrogen) according to manufacturer specifications, followed by Phenol-CHCl3 extraction and purified using Zymo RNA Clean and Concentrator columns. RNA was eluted in 30 uL ultrapure water and strand-specific RNA-seq libraries were prepared using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) and the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB #E7760S/L) and sequenced an Illumina NovaSeq X-Plus.