RNA-seq of live cell 3-D models with engineered genetic sub-types of lung cancer

An experimental system was designed to screen for targetable signaling pathways linked to early 3D invasion in molecular subtypes, TP53 and LKB1, of KRAS-driven lung adenocarcinoma (LUAD). Live-cell imaging of human bronchial epithelial cells during 3D invasion was combined with RNA transcriptome profiling shown here. Overall design: RNA-seq was performed in triplicate on for each sub-type. Total RNA was extracted from the spheroids at day 7 of 3D spheroid invasion assay using a RNeasy Kit (Qiagen, Valencia, CA, USA). The quantitation, integrity and purity of the extracted total RNA samples were assessed by the Emory Integrated Genomics Core (EIGC) using 2100 Bioanalyzer (Agilent) and RNA sequencing was performed by Novogene, Co., Ltd. Data processing, quality control, read alignment and statistical analyses were performed by the Emory Biostatistics and Bioinformatics Shared Resource, as previously described?(21)?. Raw read data (fastq) QC was performed using FastQC v0.11.7. Post-filtered reads were mapped against Ensemble Human GRCh38.p12 release 95 reference genome using the STARaligner v2.7.0e. Expression quantification was obtained using featureCounts. Normalization and pairwise differential analysis were determined using median-ratios method in DESeq2 and log2 transformed. Differentially expressed genes (DEGs) were identified using a moderated t-test as implemented in the Limma R package. Heatmaps were created by unsupervised clustering of log2 transformed normalized expression data for the significant DEGs, and the resulting data were analyzed using a Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG).