Large-Scale Single-Nucleus RNA Sequencing Compatible with Complex Archived Samples

Abstract

Transcriptome profiling at single-cell resolution allows us to identify and assess functional cell types and cellular states, including those within degenerating ocular tissues in retinitis pigmentosa. The technology is particularly valuable when studying tissues with high cellular heterogeneity, or when specific cell types are of interest. In this chapter, we introduce a detailed protocol of a medium-throughput single-nucleus RNA sequencing technique that utilizes frozen tissue as input sample. This protocol can be executed by any researcher with basic training in molecular biology techniques. With this protocol, a single experimenter can easily process two samples per day up to cDNA amplification, and library preparations can be done in batches of 8. Routinely we can obtain ~20 K nuclei per eye from 3 to 4 library preparations.

Keywords: Deep sequencing; Gene expression profiling; Parallel mRNA analysis; Single-cell transcriptomics; sNuc-seq.