Nanopore target PCR on CHRNA5 region
Cells treated with DMSO or CSC were seeded in 6-well culture for 24 hours. The cells were transfected with 2.5ug of the pET01-CHRNA5-Exon5-minigene using Lipofectamine 3000 in biological replicates, an empty-pET01 vector, and a GFP control plasmid. Media was replaced four hours post-transfection and cells were harvested for RNA extraction 48 hours post-media change. Amplicons for ONT sequencing were generated from cDNA by RT-PCR to include ONT adapter sequences. Libraries for amplicons were prepared as specified in the Ligation sequencing V14  PCR barcoding protocol (VPBC_9182_v114_revK_07Mar2023, ONT). Amplicons were multiplexed in the libraries using EXP-PCB001 kit (ONT) and pooled in equal molar amounts as calculated from fragment size and mass. The library was loaded onto a R10.4.1 PromethION flow cell (ONT) mounted on a PromethION 2 Integrated device and run for 72 hours with barcode balancing active. High accuracy basecalling was performed using MinKNOW software.