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. 2020 Sep 18;1(2):100081.
doi: 10.1016/j.xpro.2020.100081.

Miniaturization of Smart-seq2 for Single-Cell and Single-Nucleus RNA Sequencing

Baptiste N Jaeger  1 , Emilio Yángüez  2 , Lorenzo Gesuita  3 , Annina Denoth-Lippuner  1 , Merit Kruse  1 , Theofanis Karayannis  3 , Sebastian Jessberger  1
Affiliations

Miniaturization of Smart-seq2 for Single-Cell and Single-Nucleus RNA Sequencing

Baptiste N Jaeger et al. STAR Protoc. .

Abstract

This protocol presents a plate-based workflow to perform RNA sequencing analysis of single cells/nuclei using Smart-seq2. We describe (1) the dissociation procedures for cell/nucleus isolation from the mouse brain and human organoids, (2) the flow sorting of single cells/nuclei into 384-well plates, and (3) the preparation of libraries following miniaturization of the Smart-seq2 protocol using a liquid-handling robot. This pipeline allows for the reliable, high-throughput, and cost-effective preparation of mouse and human samples for full-length deep single-cell/nucleus RNA sequencing. For complete details on the use and execution of this protocol, please refer to Bowers et al. (2020).

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Schematic Representation of the Nucleus Dissociation Protocol for the Mouse Brain
Figure 2
Figure 2
Schematic Representation of the Cell Dissociation Protocol for the Neonatal Mouse Brain
Figure 3
Figure 3
Schematic Representation of the Cell Dissociation Protocol for Human Forebrain Organoids
Figure 4
Figure 4
Schematic Representation of the Key Settings Recommended for Single-Cell/Nucleus Sorting into 384-Well Plates Using the FACS Aria III (BD Biosciences) (A) Schematic representation of the stream and the nozzle to be used in the protocol. (B) Drop location in home position. (C) Recommended plate layout.
Figure 5
Figure 5
Gating Strategies to Identify Single Cells/Nuclei Prior to Sorting Representative FACS plots showing examples of gating strategies for the isolation of NeuN+ nuclei from the adult hippocampus (A), tdTom-labeled neurons (whole cells) from the postnatal mouse brain at P9 (B), and tdTom+ cells isolated from human forebrain-regionalized organoids after 36 days in culture (C).
Figure 6
Figure 6
Schematic Representation of the Main Steps of the Miniaturized Smart-seq2 Protocol
Figure 7
Figure 7
Examples of cDNA Profiles Obtained on the Bioanalyzer Instrument
Figure 8
Figure 8
Examples of Library Profiles Obtained on the TapeStation Instrument
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