Preparation of Intact Nuclei for Single-Nucleus Omics Using Frozen Cell Suspensions from Mutant Embryos ofXenopus tropicalis

Abstract

Single-cell omics such as single-cell RNA-sequencing (RNA-seq) have been used extensively to obtain single-cell genome-wide expression data. This technique can be used to compare mutant and wild-type embryos at predifferentiation stages when individual tissues are not yet formed (therefore requiring genotyping to distinguish among embryos), for example, to determine effects of mutations on developmental trajectories or congenital disease phenotypes. It is, however, hard to use single cells for this technique, because such embryos or cells would need to be frozen until genotyping is complete to capture a given developmental stage precisely, but intact cells cannot be isolated from frozen samples. We developed a protocol in which high-quality nuclei are isolated from frozen cell suspensions, allowing for genotyping individual embryos based on a small fraction of a single embryo suspension. The remaining suspension is frozen. After genotyping is complete, nuclei are isolated from embryo suspensions with the desired genotype and encapsulated in 10× Genomics barcoded gel beads for single-nucleus RNA-seq. We provide a step-by-step protocol that can be used for single transcriptomic analysis as well as single-nucleus chromatin accessibility assays such as ATAC-seq. This technique allows for high-quality high-throughput single-nucleus analysis of gene expression in genotyped embryos. This approach may also be valuable for collection of wild-type embryonic material, for example, when collecting tissue from a particular developmental stage. In addition, freezing of tissue suspensions allows precise staging of collected embryos or tissue that may be difficult to manage when collecting and processing cells from living embryos for single-cell RNA-seq.