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

• Published in: STAR protocols Vol. 1; no. 2; p. 100081
• Main Authors: Jaeger, Baptiste N., Yángüez, Emilio, Gesuita, Lorenzo, Denoth-Lippuner, Annina, Kruse, Merit, Karayannis, Theofanis, Jessberger, Sebastian
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 18.09.2020; Elsevier
• Subjects: Animals; Base Sequence - genetics; Brain - cytology; Brain - metabolism; Cell Nucleus - metabolism; Cell Separation - methods; Gene Expression - genetics; Gene Expression Profiling - methods; Gene Library; High-Throughput Nucleotide Sequencing - methods; Humans; Mice; Miniaturization; Protocol; RNA - genetics; Sequence Analysis, RNA - instrumentation; Sequence Analysis, RNA - methods; Single-Cell Analysis - instrumentation; Single-Cell Analysis - methods; Transcriptome - genetics; Whole Exome Sequencing - methods; Workflow
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2020.100081

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). [Display omitted] •Isolation of cells or nuclei from the mouse brain and human forebrain organoids•Flow sorting of single cells or nuclei into 384-well plates•Miniaturization of the Smart-seq2 protocol using a liquid-handling robot•Protocol enables high-throughput full-length single-cell/nucleus RNA sequencing 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.