GSE183590
Single-cell RNA sequencing for the identification of early-stage lung cancer biomarkers from circulating blood
To develop biomarkers for diagnosis of early-stage lung adenocarcinoma (LUAD) patients, we set up single-cell RNA sequencing (scRNA-seq) pipeline using Fluidigm C1 systems. Through the scRNA-seq pipeline, we clustered 1,441 single cells from four non-small cell lung cancer (NSCLC) cell lines (A549, H460, H1299 and Calu3) into four groups based on differential gene expression and detected over 2,400 differentially expressed genes (DEGs) from a comparison of four clusters following dimension reduction. We validated fold change values of 120 DEGs selected from three cluster comparisons (top 20 up- and top 20 down-regulated genes per cluster comparison) using qRT-PCR approach. We performed further quantitative validation of chemokine genes (CXCL1 and CXCL2 with corresponding receptor, CXCR2) at mRNA level in tumour and tumour-adjacent normal lung tissues of LUAD patients (16 female and 19 male). Next, we profiled microRNAs epigenetically regulating expression of those chemokine genes in the tumour and normal tissues as well as blood (plasma) samples from corresponding LUAD patients. Finally, we identified relatively high copy number of miR-532-5p regulating those chemokine genes in patient plasma samples. Overall design: We loaded 400 single cells per NSCLC cell lines (A549, H460, H1299 and Calu3) to chambers of high-throughput (HT) scRNA-seq integrated fluidics circuits (IFCs). We generated four hundreds of 3'-end enriched scRNA-seq libraries per NSCLC cell line using  in Fluidigm C1 systems (1,600 libraries in total). Those libraries were sequenced using illumina Nextseq 500 systems.