Promethion flow cell

Genomic libraries for the short and long nucleotide reads were prepared following the manufacturer protocols for genome sequencing (Illumina HiSeq 2500 and PromethION Flow Cell, R9.4.1, Nanopore). The Illumina HiSeq 2500 method employed the NEBNext Ultra DNA Library Preparation Kit, with the subsequent quality assessment conducted using the Agilent TapeStation. In the Nanopore approach, native barcoding of the genomic DNA (EXP-NBD114 and SQK-LSk109) was explicitly executed for the PromethION system. Post-barcoding, stringent quality checks were performed using Qubit and the Agilent TapeStation to ensure the reliability and quality of the prepared libraries.

Selected seedlings were exposed to a simulated frost treatment (−2°C) for 0 h, 2 h, and 9 h. Three samples were performed for each cultivar, and each sample contained 12 plants. Leaves at the third and fourth nodes from the tip were collected and stored at −80°C for further analysis.
RNA samples were prepared using an RNA simple Total RNA Kit (DP411, TIANGEN), RNA integrality was tested by agarose gel (LabChip GX, Agient2100), and the RNA concentration was detected using a NanoDrop 2000 (Thermo Fisher Scientific).
Library preparation was performed according to the standard protocol provided by ONT. A mass of 1 μg total RNA was prepared for cDNA libraries using the cDNA-PCR Sequencing Kit (SQK-LSK110+EXP-PCB096) protocol provided by ONT. The template-switching activity of reverse transcriptases enriches full-length cDNAs and adds defined PCR adapters directly to both ends of the first-strand cDNA, followed by cDNA PCR for 14 cycles with LongAmp Tag (NEB). The PCR products were then subjected to ONT adaptor ligation using T4 DNA ligase (NEB). Agencourt XP beads were used for DNA purification according to the ONT protocol. The final cDNA libraries were added to the PromethION Flow Cell (R9 Version, FLO-PRO002, Nanopore) and run on the PromethION platform at Biomarker Technology Company (Beijing, China).