GSM5769235: KD MoYPD1 RA6; Pyricularia oryzae; RNA-Seq
The fungal biomass was weighed, quick frozen in liquid nitrogen and stored at -80 oC freezer. Total RNA was isolated from 1 g of fungal biomass using TRIZOL reagent (Invitrogen Life Technologies, California, USA) according to the manufacturer's instructions. For removal of DNA contamination, extracted RNA was treated  with DNase as recommended by the manufacturer. Total RNA from KD mutant RA6 and WT biomass was used to generate cDNA using AmpliSeq cDNA synthesis kit as recommended by Illumina standard protocols. Total RNA was eluted in 500 µl RNase-free water and checked for its integrity and quantity on an Agilent Bioanalyzer. Purification of poly-A containing mRNA molecules was done using oligo-(dT) anchored magnetic beads (Sigma-Aldrich). 1ug of total RNA in 50uL was used as starting  material. Following purification, the RNA was fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments werre copied into first strand cDNA using reverse transcriptase and random primers. This was followed by second strand cDNAsynthesis using DNA Polymerase I and RNase H. These cDNA fragments then went through an end repair process, the addition of a single 'A' base, and then ligation of the adapters. The products were then purified and enriched with PCR of 12 cycles to create the final cDNA library. The quality of prepped libraries were checked on the BioAnalyser (Agilent Technologies) using the Agilent 1000 or High Sensitivity Chip. The libraries were then quantified by qPCR (KapaBiosystems), normalized, pooled and sequenced in the HiSeq 1000system, usually paired-end 100 cycles run.