Dissociation of neuronal culture to single cells for scRNA-seq (10x Genomics) v1

Abstract

This protocol outlines a method for dissociating a human pluripotent stem cell-derived neuronal culture to single cells for loading onto a Chromium 10x chip for single cell RNA-sequencing. This protocol makes a distinction between early neuronal progenitors and mature neuronal cultures, as additional steps and reagents are required in order to sufficiently dissociate the latter. These include: - DNase Vial (D2) - PDS Kit Papain Vial Note: In our labs, iPS cells were undergoing a 52-day long differentiation process to dopaminergic neurons (adapted from doi.org/10.1038/nature10648), and were treated under the 'mature' conditions when harvested from day 20 onward, and as 'early neuronal progenitors' on day 11. Example images of neuronal culture from our labs can be found in the Guidelines of this protocol for reference. This protocol assumes use of: - 12-well tissue culture plates (3.8 cm2 surface area per well ) for samples/wells being harvested for dissociation (see Materials). - A NucleoCounter®NC-200™ for purposes of cell counting.