Transcriptome profiling of human lung cancer cell lines with synthetic "sequin" controls

Purpose: The aim of this study is to build up a transcriptomic data analysis pipeline and test its performance using synthetic "sequins" controls and lung cancer cell lines. Methods - Cell Culture: Lung adenocarcinoma cell lines NCI-H1975 and HCC827 from a range of passages (2-4) were grown on 3 separate occasions in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal calf serum and 1% penicillin-streptomycin. Methods - RNA preparation: Total RNA was extracted using the TRIzol reagent. 6 ng of sequins were added to 1 ug of total RNA (mix A with H1975, mix B with HCC827). Methods - Library prep: the sequin+total RNA mixtures were subjected to polyA selection (NEBNext PolyA E7490) and library prep (NEBNext Ultra II Directional RNA Library Prep Kit for Illumina) following manufacturer's instructions; we performed 9 PCR cycles Overall design: Total RNA was extracted from lung adenocarcinoma cell lines NCI-H1975 and HCC827 (2 independent samples for each cell line). Both mRNA and Total RNA transcriptomes from these samples were profiled by RNA-Seq (Illumina short-read, Oxford Nanopore Technologies PromethION and PacBio Sequel II) The processed data are the raw counts of all Sequin genes. Although there are some reads from human cell lines in this dataset, by mapping all of the reads to the Sequin decoy chromosome, we can get a mapping rate around 80%, and we only analysed the Sequins genes in our study.