Sqk rna002 direct rna sequencing kit

The genome of C. lanceolata was not available due to its large size. Thus, we also used long-read sequencing based on nanopore direct RNA-seq (DRS) to generate a high quantity of unigenes and to improve the integrity of the unigenes. The RNA quality was determined using a NanoDrop 2000c UV–Vis spectrophotometer (Thermo Fisher, Waltham, MA, USA) followed by 1% agarose gel electrophoresis, and the high-quality total RNA with RIN values higher than 8 was used. The total RNAs of the secondary xylem from different stages and different treatments were mixed into one sample for DRS, and mRNA was isolated using the Dynabeads mRNA Purification Kit (AMBION, CAT#61006). The DRS library was built using the SQK-RNA002 Direct RNA Sequencing Kit (Oxford Nanopore Technologies). The constructed library was loaded onto the R.9.4.1 FlowCell (FLO-MIN106) for sequencing in a MinION MK 1B sequencer running for 48 h. After sequencing, the electronic raw signals were identified using Guppy (version 2.3.1) with the default parameters. The transcriptome data generated by DRS were corrected by lordec-correct (-k 21 -s 3) [74 (link)] using RNA-seq reads to obtain more accurate long transcripts. Finally, reference transcript sequences were generated using CD-HIT software [75 (link)] to remove redundancy.