Effect of a nsp1 deletion in SARS-CoV-2 virus; GFP; 293T
HEK293T cells in six-well plate were transfected with 2.5 g of nsp1-expressing plasmids or pcDNA3.1 control plasmid using Lipofectamine 2000 (Invitrogen, USA). Twenty-four hours after transfection, cells were harvested for total RNA extraction using miRNeasy Micro Kit (QIAGEN, Germany) according to the manufactures instruction. The concentration of RNA was measured by Qubit 3 (Invitrogen, USA), and the quality control (QC) was determined by Qsep1 (BiOptic, China) capillary gel electrophoresis (CGE) with an R1 RNA cartridge. RNA-Seq was performed using nanopore MinION platform (Oxford Nanopore Technologies, UK) Direct cDNA Sequencing Kit (SQK-DCS109) with the Native Barcoding Expansion 1-12 (EXP-NBD104) according to the manufactures instruction. Five-hundred nanogram of total RNA for each samples were used as input RNA. After barcoding, an aliquot of each samples was preserved for cDNA fragment length determination using Qsep1 CGE with a S2 DNA cartridge. When pooling barcoded samples, molarities were calculated from mass concentration using Biomath Calculators to make sure that the total library loaded on flow cell would not exceed 200 fmol.