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.

提取授粉后16天（DAP）的胚乳（endosperm）组织中的总RNA。以寡聚(dT)30锚定引物合成第一链cDNA，并通过长距离PCR（LD-PCR）扩增获得双链cDNA。将所得cDNA按片段大小分选，构建1-2 Kb、2-3 Kb及3-6 Kb三个文库。依照PacBio RSII Iso-Seq实验方案，对每个文库开展异构体测序（isoform sequencing，Iso-Seq）。所得原始数据将被转换为.h5格式，随后通过SMRT软件包（SMRT Package）Iso-Seq流程的默认参数，处理为插入序列读段（read of insert，ROI）。