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. 2020 Sep 20;10(18):e3749.
doi: 10.21769/BioProtoc.3749.

Probe-Seq: Method for RNA Sequencing of Specific Cell Types from Animal Tissue

Ryoji Amamoto  1 , Mauricio D Garcia  1 , Emma R West  1 , Jiho Choi  1 , Sylvain W Lapan  1 , Elizabeth A Lane  2 , Norbert Perrimon  2 , Constance L Cepko  1
Affiliations

Probe-Seq: Method for RNA Sequencing of Specific Cell Types from Animal Tissue

Ryoji Amamoto et al. Bio Protoc. .

Abstract

Most organs and tissues are composed of many types of cells. To characterize cellular state, various transcription profiling approaches are currently available, including whole-tissue bulk RNA sequencing, single cell RNA sequencing (scRNA-Seq), and cell type-specific RNA sequencing. What is missing in this repertoire is a simple, versatile method for bulk transcriptional profiling of cell types for which cell type-specific genetic markers or antibodies are not readily available. We therefore developed Probe-Seq, which uses hybridization of gene-specific probes to RNA markers for isolation of specific types of cells, to enable downstream FACS isolation and bulk RNA sequencing. We show that this method can enable isolation and profiling of specific cell types from mouse retina, frozen human retina, Drosophila midgut, and developing chick retina, suggesting that it is likely useful for most organisms.

Keywords: Cell Types; FISH; RNA; RNA-sequencing; Transcriptional Profiling.

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Conflict of interest statement

Competing interestsThe authors declare no conflict of interest.

Figures

Figure 1.
Figure 1.. Flow Chart for Probe-Seq protocol
Figure 2.
Figure 2.. Example DNA gel of three gene-specific probe sets.
The left lane is the DNA ladder with the 500 bp band indicated in red. For each lane, the bottom band is the unextended hairpin and other small DNA pieces. The top band is the extended probe set. The bands are always fuzzy because the probes are ssDNA.
Figure 3.
Figure 3.. FACS gating strategy for Probe-Seq.
A. Using a Hoeschst histogram, single cell peak is gated first. B. The percentage of single cell events will depend on the tissue. For example, the mouse retina will have a higher percentage of single cell events compared to the mouse brain because less debris is generated during the dissociation protocol for the retina. Out of the single cell events, the population of interest is identified. Here, the Vsx2 fluorescence is indicated on the x-axis, and the Vsx2+ population is right-shifted from the negative population. It is advisable to use an empty autofluorescence channel as the opposing axis as this will help to determine the events that have high fluorescence in all channels. * Do not collect these events even if they have a high fluorescence intensity in the Vsx2 channel. These events are also highly fluorescent for the empty autofluorescence (561 nm) channel. If using a histogram or a plot without an autofluorescence channel, these events will look like they are Vsx2+ even though they are unlikely to be the population of interest.
See this image and copyright information in PMC

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