A versatile nuclei extraction protocol for single nucleus sequencing in non model species – optimization in various Atlantic salmon tissues. v2

Abstract

Single cell RNA sequencing has rapidly become a standard tool for profiling transcriptomic diversity across thousands of cells (Linnarsson and Teichmann, 2016), and is now being applied to a large diversity of species and tissues. The main limitation of this technology is that it requires the isolation of live cells from fresh tissue, severely restricting its applicability. As a result, single nuclei RNA sequencing (snRNA-seq), which consists of sequencing the RNA of only the nuclei of cells rather than of the whole cell, has been commonly adopted since it allows samples to be stored for several months prior to processing while yielding comparable results to whole cell sequencing (Kulkarni, et al., 2019; Slyper et al. 2021). A critical challenge for snRNA-seq is the successful extraction of high quality nuclei. This has spurred the recent publication of a number of dissociation protocols for nuclei extraction (Drokhlyansky et al. 2020; Eraslan et al. 2021; Melms et al 2021), however, these have largely been optimized for model species such as humans, and more and more single nuclei is being adopted in non-model species. Here we present a robust protocol that enables the extraction of nuclei from frozen tissue adapted from those shown to work in different tissue types, such as human skin (Drokhlyansky et al. 2020; Eraslan et al. 2021; Melms et al 2021). Our protocol has been used to successfully extract nuclei from an array of different Atlantic salmon (Salmo salar) tissues including liver, skin, fin, spleen, head kidney and gill as well as in other species such as sole (Solea solea) nose and gonad, rabbit (Oryctolagus cuniculus) nasal tissue and nurse shark (Ginglymostoma cirratum) spleen. We present the protocol as applied to fin and skin as these are particularly challenging tissues to work with given their toughness and the presence of hard tissue (e.g., scales and bones), connective tissue and fat deposits. We include notes throughout the protocol so that the reader can optimise it for a variety of tissue types. While the protocol has been optimised to work with the Chromium 10x platform, the most commonly used high throughput microfluidic device, but can be used successfully for the extraction of nuclei for other platforms and applications. The aim of this protocol is to capture 7,000 nuclei per single-nuclei RNA sequencing library using the Chromium Single Cell 3’ Reagent Kits v2 or v3 (10X Genomics). Given its utility for isolating nuclei from difficult to dissociate tissue types, we anticipate that this protocol will be broadly applicable for snRNA-seq of non-model organisms and unconventional tissue types. Thelast stepcontains a supplemental video with extra context and tips, as part of the protocols.io Spotlight series, featuring conversations with protocol authors.