PacBio RS II sequencing

The total RNA was isolated from the tissue of endosperm of 16 days after pollination (DAP). The first-strand cDNA was synthesized using with an anchored primer of oligo(dT)30, and double-strand cDNA was amplified by long-distance PCR (LD-PCR). The resulting cDNA was size-selected for three libraries of 1-2 Kb, 2-3 Kb and 3-6 Kb. Each library was subjected to the isoform sequencing (Iso-Seq) according to the PacBio RSII Iso-Seq protocol. The resulting raw data was converted into .h5 format and then converted into ROI (read of insert) reads using default parameters of the SMRT Package Iso-Seq pipeline.