RNAseq of Phakopsora pachyrhizi
PacBio Iso-Seq library was prepared according to the Iso-Seq protocol using Clontech SMARTer PCR cDNA synthesis kit (Takara Bio USA, Inc.) with no size selection. In brief, 161ng of total RNA was reverse transcribed to synthesize first-strand cDNA. The first-strand cDNA was amplified for 12 PCR cycles using KAPA HiFi DNA polymerase (Kappa Biosystems). The amplified DNA was subjected to damage and end-repair (SMRTbell Damage Repair Kit; Pacific Biosciences), and then ligated to a SMRTbell adapter for Iso-Seq SMRTbell library construction. A sequencing primer was annealed to SMRTbell templates (SMRTbell Template Prep Kit 1.0; Pacific Biosciences) which was then bound to DNA polymerase (DNA/Polymerase Binding Kit P6 v2; Pacific Biosciences) and prepared for Magbead loading (Magbeads, Pacific Biosciences) onto SMRT cells. Sequencing was performed on a PacBio RS II system using P6 polymerase and chemistry (DNA Sequencing Reagent 4.0 v2; Pacific Biosciences) and a 360-min movie time. Final library composed of transcripts with no size selection was sequenced on six SMRT cells.
10Dai_S2-ns_S4-ns_052617_39/A01_4