MinION sequencing; Nanopore RNA-seq of human osteosarcoma cell line U2OS treated with siRNA against MDC1, PLRG1, or control siRNA with or without pladienolide B
Nanopore RNA-seq of human osteosarcoma cell line U2OS treated with siRNA against MDC1, PLRG1, or control siRNA with or without pladienolide B
Total RNA was extracted from U2OS cells 72 hours after siRNA transfection and 3 hours following treatment with pladienolide B inhibitor (5 nM) (3 independent biological replicates per condition). U2OS cells were treated with siRNA-mediated depletion of MDC1 and PLRG1, or treated with the splicing inhibitor pladienolide B (5 nM). Cells were treated with control siRNA as control condition. Total RNA extraction was carried out using RNeasy Mini Kit (QIAGEN) according to manufacturerâ€™s instructions. Oxford Nanopore Technologies (ONT) kits SQK-PCS109 and SQK-PBK004 were used for preparation of the barcoded cDNA library according to the manufacturer. The initial input was 70 ng of total RNA per sample. cDNAs were amplified using 18 PCR cycles.