Abstract
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. Here, we 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). We 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.
Highlights
Multicellular eukaryotic tissues often comprise many different cell types, commonly classified using their morphological features, physiological functions, anatomical locations, and/or molecular markers
To determine whether Probe-Seq can enable the isolation and profiling of specific cell types based upon fluorescent in situ hybridization (FISH) labeling, we tested it using the mouse retina
We have shown that FGF8, CYP26C1, and CYP26A1 are highly enriched in this area at embryonic day 6 (E6), the other molecular determinants that may play a role in high acuity area development are unknown
Summary
Multicellular eukaryotic tissues often comprise many different cell types, commonly classified using their morphological features, physiological functions, anatomical locations, and/or molecular markers. ScRNA profiling technologies have led to the appreciation of many subtypes of these major cell classes, bringing the total number of retinal cell types close to 100 (Macosko et al, 2015; Rheaume et al, 2018; Shekhar et al, 2016). Such accelerated discovery of cellular diversity is not unique to the retina, as scRNA profiling is being carried out in many tissues and organisms (Tabula Muris Consortium et al, 2018; Han et al, 2018; Regev et al, 2017).
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