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

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

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).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence / genetics
  • Brain / cytology
  • Brain / metabolism
  • Cell Nucleus / metabolism
  • Cell Separation / methods
  • Exome Sequencing / methods
  • Gene Expression / genetics
  • Gene Expression Profiling / methods
  • Gene Library
  • High-Throughput Nucleotide Sequencing / methods
  • Humans
  • Mice
  • Miniaturization
  • RNA / genetics
  • Sequence Analysis, RNA / instrumentation*
  • Sequence Analysis, RNA / methods
  • Single-Cell Analysis / instrumentation*
  • Single-Cell Analysis / methods*
  • Transcriptome / genetics
  • Workflow

Substances

  • RNA