ONT cDNA-seq of Arabidopsis thaliana: mature leaf, biological replicate 2
For ONT libraries RNA was converted to cDNA using Mint cDNA synthesis kit (Evrogen, Moscow, Russia) with 18 cycles of amplification with the following modification: 1) cDNA synthesis is primed with oligonucleotides that contain not only dT part and adapter part but also custom barcode, specific for each sample; 2) optimal number of cycles for amplification was selected based on the results of qPCR with primers annealing at 3 and 5 ends. The number of cycles falling to the upper part of linear phase was considered to be optimal. Amplified cDNA was used as input for library preparation using standard protocol for genomic DNA with LSK-109 kit. Libraries were sequenced on PromethION instrument using R9.4.1 flowcell.