The development of a large variety of single-cell analytical methods has empowered researchers to explore diverse biological questions at the level of individual cells. Among these, droplet-based single-cell RNA sequencing (scRNA-seq) methods have been particularly prevalent owning to their high-throughput capabilities and reduced reaction volumes. While commercial systems have contributed to the widespread adoption of droplet-based scRNA-seq, the relatively high cost impose limitations for profiling large numbers of samples. Moreover, as the scope and scale of single cell sequencing methods keeps expanding, the possibility to accommodate diverse molecular biology workflows and inexpensively profile multiple biospecimens simultaneously, becomes highly relevant. Herein, we present inDrops-2: an open-source scRNA-seq platform designed to profile fresh or preserved clinical samples with a sensitivity matching that of state-of-the-art commercial systems, yet at the few folds lower cost. Using inDrops-2, we conducted a comparative analysis of two prominent scRNA-seq protocols - those based on exponential and linear amplification of cDNA - and provide new insights about the technical biases inherited to each approach. Finally, we applied inDrops-2 to simultaneously profile 18 lung carcinoma samples, all in one run following cell dehydration, long-term storage and multiplexing, to obtain a multiregional cellular profile of tumor microenvironment; thus, inDrops-2 offers researchers a possibility to perform large scale transcriptomics studies in a cost-effective manner.

inDrops-2: a flexible, versatile and cost-efficient droplet microfluidics approach for high-throughput scRNA-seq of fresh and preserved clinical samples