title: GSM7509500: scRNA-seq 2; Homo sapiens; RNA-Seq
library_construction_protocol: Cancerous epithelial cells (n=3,754), as determined as those cells harboring large chromosomal aberrations from InferCNV analysis, were subset and re-clustered using the following parameters: Ngenes = 2,000, Npcs = 20, Res = 0.9, Kparam = 10 (script NI04). We found the differences in gene expression between the three treatment time points (TN, RD, and PD) using the Seurat function Find-Markers using the MAST test21 and sample_name as the latent variable. Three separate tests were used to ascertain the differences between: 1) TN and RD, 2) TN and PD and 3) RD and PD (TableS5). Resulting differential gene lists were then filtered to limit patient specific effects. This is achieved by setting a threshold for non-zero expressing cells per patient (RD = 3 of RD patients and PD = 6 of PD patients) and removing differentially expressed genes explained by less than the thresholds set. The top 100 genes from each comparison were manually curated to evaluate for pathway activation. Decreased expression could indicate lack of detection due to the stochasticity of scRNaseq and thus for analysis of activated pathways we focused on upregulated genes. Gene signatures (TableS2) were compiled using differential expressed as well as known cell marker genes. Specifically, the alveolar signature is made of differentially expressed AT1/AT2 genes among the cancer cell time point comparisons as well has additional known AT1/AT2 genes 22. The remaining signatures were identified directly from top differentially expressed genes. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics).