title: Illumina MiSeq paired end sequencing; Saccharomyces cerevisiae transcriptome response to overexpression of the Phytophthora sojae Avh172 effector
design: Saccharomyces cerevisiae transcriptome response to overexpression of the Phytophthora sojae Avh172 effector
library_construction_protocol: Portions of each culture (1.5 mL) was transferred to a microcentrifuge tube and centrifuged at 6000x g for three minutes. Supernatant was discarded and pellet was frozen at -80°C until ready for RNA isolation procedure.  BY4741 yeast transformed with the yeast expression constructs pAG425GAL-PsAvh172 (experimental) or pAG425GAL-ΔGFP (control) were inoculated into quadruplicate tubes containing 4 mL of non-inducing selective synthetic media supplemented with 2% raffinose (SC-LEU + 2%Raf). Following overnight growth, saturated cultures were sub-inoculated to non-inducing selective synthetic media supplemented with 2% raffinose to OD595 0.25 and incubated at 30°C for two hours. Raffinose cultures were supplemented with 20% galactose to 2% final concentration and incubated at 30°C for an additional two hours.  RNA isolation and DNase treatment was performed using the MasterPure Yeast RNA Purification Kit (Epicentre) according to the manufacturer's instructions, with final RNA re-suspension in 25 μL TE Buffer. RNA was quantified using the Thermo Scientific NanoDrop 1000 and Invitrogen QUBIT 2.0 Flourometer. RNA quality was analyzed using the Agilent BioAnalyzer RNA6000. The three most high quality replicates, as determined by visual inspection of Agilent BioAnalyzer RNA6000 electropherogram, of each transformant (pAG425GAL-PsAvh172 and pAG425GAL-ΔGFP) were chosen for RNAseq transcriptome analysis. RNA TruSeq libraries were prepared for sequencing using standard Illumina protocols