title: MinION sequencing; Nanopore RNA-seq of human cell lines treated with or without nicotinamide adenine dinucleotide (NAD+)
design: Nanopore RNA-seq of human cell lines treated with or without nicotinamide adenine dinucleotide (NAD+)
library_construction_protocol: Total RNA was extracted from cells after treatment with or without NAD+ (3 biological replicates per cell line per condition). Cells were treated for 24 hours with 2 mM NAD+. Cells were untreated as the control condition. Total RNA was extracted with the RNeasy Mini Kit (QIAGEN) according to the manufacturer's instructions, including QIAshredder spin columns for homogenization. Total RNA was used as input with the library preparation SQK-PCB109 kit (Oxford Nanopore Technologies, ONT) according to manufacturer's instructions. Two libraries were prepared: one with HeLa and U2OS samples, and one with BJ and BJ-5ta samples. In the barcoding-PCR step, 14 cycles with 4 min extension time were performed. Barcoded cDNA samples were pooled into a library with a final mass of 100 ng.