Single molecule RNA sequencing (Iso-seq) experiment and data
analysis
For gene annotation of the genome, transcriptome sequencing was
performed with mixed tissues of a young seedling (14 day after
imbibition). RNA was extracted with the TRIzol Reagent (Invitrogen,
USA). The RNA quality was checked by a spectrophotometer (LabTech, USA)
and a 2100 Bioanalyzer (Agilent Technologies, USA). The verified RNA was
used for transcriptome sequencing library construction. Briefly, the
mRNA was reversely transcribed using a Clontech SMARTer cDNA synthesis
kit. A BluePippin Size Selection System (Pacific Biosciences of
California, Menlo Park, CA, USA) was used to perform the size selection
for the two libraries, sized 0–3 kb and 2–6 kb, respectively, after
cDNA amplification and purification. The SMRTbell libraries were
constructed according to the manufacturer’s protocol, and sequenced on
the PacBio SEQUEL II platform (Pacific Biosciences of California, Menlo
Park, CA, USA). Last, we used SMRTLink 7.0
(https://www.pacb.com/support/software-downloads/) to produce all
the mRNA sequences for genome annotation.