I002C ONT cDNA
Total RNA was extracted from the I002C lymphoblastoid cell line using the Bioline Isolate II RNA Mini Kit (Meridian Biosciences) according to manufacturer's instructions. Total RNA quantification, purity and integrity assessment was performed using the Qubit 3.0 Fluorometer and Qubit BR RNA Assay Kit (ThermoFisher Scientific), NanoDrop 2000 spectrophotometer (ThermoFisher Scientific) and Agilent 4200 TapeStation system with the RNA screentape assay kit (Agilent). 39g of total RNA was brought forward to poly(A)+ RNA enrichment using the Dynabeads mRNA Purification Kit (ThermoFisher Scientific) according to manufacturers instructions. Nanopore cDNA-PCR library was generated from 50ng of poly(A)+ RNA using the SQK-PCB114.24 kit, (Oxford Nanopore Technologies) according to manufacturers instructions. Briefly, complementary strand synthesis and strand switching were performed on input full-length poly(A)+ RNA using kit-supplied oligonucleotides, 16-cycle PCR amplification using primers containing 5 tags to generate a double-stranded cDNA library, followed by ligase-free attachment of rapid sequencing adaptors. Final cDNA library was loaded onto a FLO-PRO114M R10.4.1 flowcell (Oxford Nanopore Technologies) and sequenced on a PromethION P24 A100 instrument, PromethION release version 25.03.7, Dorado server 7.8.3, equivalent to Dorado release 0.9.2, with live-basecalling in super-accurate (SUP) mode and barcoding trimming.