GridION sequencing of SAMD00827016
Plant samples were frozen with liquid nitrogen, and crushed using a TissueLyser II (QIAGEN). RNA was purified using the Invitrogen Plant RNA Reagent and the Direct-zol RNA Kit (Zymo Research, R2052). Purified RNA was used for library preparation with the PCR-cDNA Barcoding kit (Nanopore, SQK-PCB109). Reverse transcription was performed using Maxima H Minus Reverse Transcriptase (Thermo Scientific), and PCR amplification was carried out for 14-16 cycles using LongAmp Taq 2X Master Mix (New England Biolabs) with Barcode primers (BP01-11). Subsequently, the samples were treated with Exonuclease I (New England Biolabs), purified with AMPure XP beads (Beckman), and combined to construct libraries containing a total of 100 fmol per run.