Long-read transcriptome profiling of human lung cancer cell lines
Purpose: The aim of this study is to compare the transcriptome profiles using a mixture design that allows the relative changes of the majority of the genes profiled to be estimated.   Methods - Cell Culture: Lung adenocarcinoma cell lines NCI-H1975 and HCC827 were grown in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal calf serum and 1% penicillin-streptomycin.  Methods - RNA preparation: mRNA was extracted using a Qiagen RNA miniprep kit and purified using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (E7490).  Purified mRNA spiked with sequins was used for the cRNA-PCR Barcoding (SQK-PCS109 with SQK-PBK004) kit (ONT). Completed libraries were sequenced on PromethION (ONT). Reads were mapped to known genomic features of the GrCh38 reference transcriptome and RNA sequin transcriptome combined sequences at the transcript-level and single reads were then summarized into transcript-level counts using salmon software (Patro et al. 2017). Overall design: Total RNA was extracted from lung adenocarcinoma cell lines NCI-H1975 and HCC827. Total RNA transcriptome from these samples was profiled by RNA-seq (Oxford Nanopore Technologies PromethION).