title: Illumina HiSeq 2000 paired end sequencing; Analysis of the effect of the farnesol on transcript levels and polysome association in C. albicans
design: Analysis of the effect of the farnesol on transcript levels and polysome association in C. albicans
library_construction_protocol: Grown in YPD media at 37oC to exponential phase (OD600 0.5-0.7), pelleted by centrifugation and snap frozen into liquid nitrogen.  Some samples were treated with 100uM farnesol for 15minutes before RNA extraction. Samples lysed in Lola lysis buffer using a 6870 Freezer Mill (Spex). Lysates cleared by centrifuging twice at 15000g.  For polysome associated samples 10A260 units were loaded onto a polysome gradient, fractionated then polysome fractions pooled. RNA was extracted from polysome associated and Total samples using Trizol Reagent followed by isopropanol precipitation. rRNA was depleted from samples using the Ribo-Zeroâ˘ Magnetic Gold Kit (Yeast) for RNA-Seq (Epicentre, Madison). Depleted samples were ethanol precipitated and re-suspended in 10 ď­l DEPC water.  Sequencing libraries were generated using the TruSeq Stranded mRNA sample preparation kit from Illumina.