title: RNA-seq of S. cerevisiae (BY4741) treated with Lanosterol against untreated controls in the presence and absence of alpha-factor
design: RNA-seq of S. cerevisiae (BY4741) treated with Lanosterol against untreated controls in the presence and absence of alpha-factor
library_construction_protocol: The yeast culture was treated and then centrifuged. Yeast cells (BY4741) were grown at 30°C at 200 r.p.m. in YPD medium. The cells were grown in the presence or absence of lanosterol (LST) at a final concentration of 1 μM. Post-metabolite loading, the cells were either treated or untreated with the α-factor at a final concentration of 30 μM for 2 hours. RNA isolation was done using TRIzol Reagent. The sample was homogenized with TRIzol Reagent, followed by addition of chloroform. The solution was allowed to separate into different layers, and the upper aqueous layer was transferred to isopropanol, in the presence of glycogen for precipitation. The precipitated RNA was washed with ethanol, and resuspended in nuclease-free water. DNAse treatment was done to remove DNA contamination. The pure RNA was again precipitated with ethanol, washed and resuspended in nuclease free water for downstream processing. the nucleic acid library was successfully prepared