P. infestans US24 Isolate #2, No previous mefenoxam exposure, biological replicate 2
Strand-specific RNA sequencing, following the method of Zhong et al. (2011) was used to examine gene expression differences between non-exposed isolates, and those subcultures of the same isolates after acquisition of resistance to mefenoxam.    There were two treatments used for each isolate; in the first treatment, the isolate was cultured on pea agar without mefenoxam, and in the second treatment, the isolate was cultured on pea agar containing 100 μg ml-1 mefenoxam. These treatments were continued for three successive subcultures. Subsequently, those individuals that had been grown on mefenoxam-free medium were transferred to pea broth free of mefenoxam and those individuals grown on pea medium with mefenoxam were transferred to pea broth with mefenoxam (100 μg ml-1), after which all individuals were harvested for RNA extraction.      Each isolate and treatment combination was replicated three times for three separate biological replicates.    Within each biological replicate there were two pooled technical replicates, comprised of two individuals cultured synchronously as described above. Total RNA was extracted using the RNeasy Plus Mini kit (QIAGEN). Libraries were prepared following the method described in Zhonget al.(2011), and 20 ng of each library was were pooled multiplexed into a single sample and sequenced run on an Ilumina HiSeq 2000 via 100-bp single-end read sequencing in a single lane at the Cornell University Sequencing Core Facility.    RNA-Seq reads were first aligned to ribosomal RNA and tRNA sequences using Bowtie and allowing for two mismatches to remove any possible contaminations of these sequences. The resulting filtered reads were aligned to the draft genome of P. infestans strain T30-4, available from the Broad Institute (Phytophthora infestans Sequencing Project, Broad Institute of Harvard and MIT, http://www.broadinstitute.org) using TopHat allowing one segment mismatch.