Full-length cDNA sequencing of PT-06
We isolated total RNA from leaves using the Monarch Total RNA Miniprep Kit (New England Biolabs, United Kingdom). The 6 l reverse transcription reaction contained 3 l of ~200 ng total RNA, 2 l of 10 M Reverse Transcription Primer (5'AGCAGTGGTATCAACGCAGAGTAC(T)30V3') and 1 l of 10 mM dNTP. The reaction was incubated at 70C for 5 min and held on ice to allow the primer to anneal to mRNA with poly(A) tail. cDNA synthesis was performed by adding 2.5 l of Template Switching RT Buffer, 1 l of Template Switching RT Enzyme Mix (#M0466, New England Biolabs, United Kingdom), and 0.5 l of 75 M Template Switching Oligo (TSO) (5'GCTAATCATTGCAAGCAGTGGTATCAACGCAGAGTACATrGrGrG3') to the 6 l primer annealed reaction. The reaction was incubated at 42C for 90 min, 85C for 5 min, and held at 4C. Full length cDNA (fl cDNA) was amplified by adding 12.5 l of Q5 Hot Start High Fidelity 2X Master Mix (New England Biolabs, United Kingdom), 10 l of nuclease free water, and 1 l of 10 M cDNA PCR Primer (5'AAGCAGTGGTATCAACGCAGAGT3') to the 10 l cDNA product. The thermal cycling profile was 98C 45 s, 20 [98C 10 s, 62C 15 s, 72C 3 min], 72C 5 min, for yielding ~1 g fl cDNA product. We cleaned up the fl cDNA using 1.2 AMPure XP (Beckman Coulter, United States). Nanopore libraries were constructed with the ligation sequencing chemistry using ~200 fmol pooled library (~250 ng for 2,000 bp cDNA). Nanopore libraries were then sequenced and basecalled using the super accuracy model on a GridION system (Oxford Nanopore Technologies, United Kingdom) at the Department of Biology, University of Oxford. Basecalled reads were trimmed for Nanopore adaptors, the primer sequences, and split for chimeras using dorado 0.6.2+14a7067.