Probe-Seq – transcriptional profiling of specific cell types from heterogeneous tissue by RNA-based isolation

Recent transcriptional profiling technologies are uncovering previously-undefined cell populations and molecular markers at an unprecedented pace. While single cell RNA (scRNA) sequencing is an attractive approach for unbiased transcriptional profiling of all cell types, a complementary method to isolate and sequence specific cell populations from heterogeneous tissue remains challenging.

Harvard Medical School researchers have developed Probe-Seq, which allows deep transcriptional profiling of specific cell types isolated using RNA as the defining feature. Dissociated cells are labeled using fluorescent in situ hybridization (FISH) for RNA, and then isolated by fluorescent activated cell sorting (FACS). The researchers used Probe-Seq to purify and profile specific cell types from mouse, human, and chick retinas, as well as from Drosophila midguts. Probe-Seq is compatible with frozen nuclei, making cell types within archival tissue immediately accessible. As it can be multiplexed, combinations of markers can be used to create specificity. Multiplexing also allows for the isolation of multiple cell types from one cell preparation. Probe-Seq should enable RNA profiling of specific cell types from any organism.

Isolation and transcriptional profiling of specific BC subtypes from the adult mouse retina

rna-seq

(a) Schematic of Probe-Seq for the adult mouse retina. The retina was dissociated into single cells, fixed, and permeabilized. Cells were incubated with gene-specific probe sets against Vsx2 and Grik1 and subsequently incubated with fluorescent oligos. Three populations of labeled cells (Vsx2, Vsx2+/Grik1, and Vsx2+/Grik1+) were isolated by FACS for downstream RNA sequencing. (b) SABER-FISH signals from an adult mouse retina section using Vsx2 and Grik1 probe sets. (c) Representative FACS plot of all events (top panel) on a Hoechst histogram.  (d) Expected distribution of retinal cell type markers expressed in each isolated population. (e) Images of dissociated mouse retinal cells after the SABER-FISH protocol on dissociated mouse retinal cells before FACS. (f) A heatmap representing relative expression levels of BC subtype markers previously identified by scRNA sequencing that are differentially expressed (adjusted p-value<0.05) between Grik1 and Grik1+ populations. (g) A representative scatter plot of log2 normalized counts of all genes (left panel) or BC2 – BC4 marker genes (right panel) between Live Grik1+ cells and Probe-Seq Grik1+ Cells. (h) A heatmap of the stain index with varying levels of transcript expression and number of oligos. (i) Schematic and flow cytometry plots of iterative Probe-Seq.

Amamoto R, Garcia MD, West ER, Choi J, Lapan SW, Lane EA, Perrimon N, Cepko CL. (2019) Probe-Seq enables transcriptional profiling of specific cell types from heterogeneous tissue by RNA-based isolation. Elife [article]

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