Isolation and Sequencing of Full-Length cDNA

Total RNA from each sample was separately extracted and evaluated according to Liu’s method [56 (link)]. Briefly, total RNA from each sample was extracted using the Plant RNA kit RN38 EASYSpin plus (Aidlab Biotech, Beijing, China). The concentration and integrity of the total RNA was detected using an Ultraspec TM 2100 Pro UV/visible spectrophotometer and an Agilent 2100 Bioanalyzer. High-quality RNA samples were used to construct cDNA libraries. One microgram quantities of RNA from each sample were pooled together, and full-length cDNA was synthesized using the SMARTer™ PCR cDNA Synthesis Kit. The sizes of the full-length cDNAs were selected using BluePippin (SageScience, Beverly, MA, USA), and three libraries of differently sized cDNA (1–2 kb, 2–3 kb, and > 3 kb) were built. The size distribution of cDNA was then quantified using a Qubitfluorometer (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), and the quality of the three libraries was assessed using the Agilent 2100 Bioanalyzer. Subsequently, SMRT sequencing was carried out using a Pacific Biosciences RS II (Menlo Park, CA, USA) platform at Biomarker Technologies Corporation, Beijing, China.