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For Iso-sequencing, seven tissues, including 2 day-after germination seeds, 2-week-old seedling, root, stem, young leaf, mature leaf, pollinated panicles and panicles, were harvested for RNA isolation. Equal amounts of total RNA from each tissue were pooled together to identify as many isoforms as possible. SMRT bell libraries were prepared according to the Iso-Seq protocol (Isoform Sequencing (Iso-Seq™) using the Clontech SMARTer PCR cDNA Synthesis Kit and the BluePippin™ Size Selection System). The first cDNA strand was synthesized using SMARTer™ PCR cDNA Synthesis Kit (Takara Biotechnology, Dalian, China). After cycle optimization, large-scale PCR was performed to generate double-strand cDNA for size selection on the BluePippin System (Sage Science, Inc., Beverly, MA, USA). Then, another large-scale PCR was performed using the eluted DNA to generate more double-stranded cDNA. Re-amplified cDNA was purified, repaired and ligated with hairpin adapters. To minimize the bias that favors sequencing of shorter transcripts, multiple size-fractionated libraries (0-1, 0.5-1, 1-2, 1-3, 2-3 and 2-8 kb) were constructed according to the manufacture’s instruction. |
FL-cDNA |
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size fractionation |
SINGLE
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