title: SMRTseq from grass pea:leaf tissue
design: Total RNA from five-weeks old grass pea plants was isolated using RNeasy plant mini kit and residual DNA was removed with RNase-free DNase I. RNA was quantified using a Qubit 3 fluorometer and an RNA broad-range kit (Invitrogen). RNA integrity was assessed using an Agilent 2100 Bioanalyzer. Only RNA with an integrity number (RIN) greater than 8 was used for SMRTbell library preparation. An Iso-Seq library was prepared according to the Isoform sequencing protocol, using the Clontech SMARTer PCR cDNA Synthesis kit as described by Pacific Biosciences with the following modifications: One microgram of RNA, combined from two different tissues, was used as input in the Clontech SMARTer reaction. PCR cycle optimization was performed by amplification of first-strand cDNA with 10 to 18 cycles using PrimeSTAR GXL polymerase (Takara). Large-scale PCR was performed using 11 PCR cycles to generate double-stranded cDNA for SMRTbell library construction. The amplified cDNA was used to build SMRTbell template libraries according to the Iso-Seq protocol. One SMRT cell was sequenced using the PacBio Sequel platform.