Dissociation of chick embryonic tissue for FACS and preparation of isolated cells for genome-wide downstream assays

Abstract

In order to process samples by fluorescence-activated cell sorting (FACS), it is essential to obtain a single-cell suspension of dissociated cells. Numerous protocols and commercial reagents are available; however, each requires optimization for specific tissue types. Here, we describe an optimized protocol for dissociating dissected chick embryos across a broad span of developmental stages. We also provide protocols for processing targeted cell populations isolated using FACS for ATAC-seq, RNA-seq, and chromatin immunoprecipitation. For complete details on the use and execution of this protocol, please refer to Ling and Sauka-Spengler (2019) and Williams et al. (2019).

Keywords: Genomics; Model Organisms; Molecular Biology; Sequencing; Single Cell.

Graphical abstract

Figure 1

Gating plots for collecting GFP-positive and -negative cells (A) Control sample, GFP-negative, derived from non-electroporated chick embryos. (B) Experimental sample, GFP positive (green) and negative (magenta) cells isolated from chick embryos electroporated with neural crest enhancer, NC1, driving GFP.

Figure 2

Examples of ATAC profiles from Agilent TapeStation (A) TapeStation HS D1000 ladder. (B) Appropriate tagmentation yields multiple peaks of accessible chromatin at approximately 150 bp intervals. (C) Under-tagmentation results in predominantly large (>1 kb) fragments of chromatin. (D) Conversely, over-tagmentation results in predominantly small (~200 bp) pieces of chromatin.