RNA-Seq of Pyropia yezoensis: pure strain NA blades
At first, total RNA was extracted.mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Then mRNA was broken into short fragments.using mRNA as template, first strand cDNA was synthesized with random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities.After adenylation of 3  ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system. Then USER Enzyme (NEB, USA) was used with size-selected before PCR. PCR was performed and the products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina). After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform and 125 bp/150 bp paired-end reads were generated.