Repli-seq Sample Preparation using Cell Sorting with Cell-Permeant Dyes

Replication timing is significantly correlated with gene expression and chromatin organization, changes dynamically during cell differentiation, and is altered in diseased states. Genome-wide analysis of replication timing is performed in actively replicating cells by Repli-seq. Current methods for Repli-seq require cells to be fixed in large numbers. This is a barrier for sample types that are sensitive to fixation or are in very limited numbers. In this article, we outline optimized methods to process live cells and intact nuclei for Repli-seq. Our protocol enables the processing of a smaller number of cells per sample and reduces processing time and sample loss while obtaining high-quality data. Further, for samples that tend to form clumps and are difficult to dissociate into a single-cell suspension, we also outline methods for isolation, staining, and processing of nuclei for Repli-seq. The Repli-seq data obtained from live cells and intact nuclei are comparable to those obtained from the standard protocols. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Live cell isolation and staining Alternate Protocol: Nuclei isolation and staining.