Collection of cells for single-cell RNA sequencing using high-resolution fluorescence microscopy

Hendrika A Segeren; Kiki C Andree; Lisa Oomens; Bart Westendorp

doi:10.1016/j.xpro.2021.100718

Collection of cells for single-cell RNA sequencing using high-resolution fluorescence microscopy

STAR Protoc. 2021 Aug 5;2(3):100718. doi: 10.1016/j.xpro.2021.100718. eCollection 2021 Sep 17.

Authors

Hendrika A Segeren¹, Kiki C Andree², Lisa Oomens², Bart Westendorp¹

Affiliations

¹ Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands.
² VYCAP B.V., Enschede, the Netherlands.

Abstract

FACS sorting followed by single-cell RNA-sequencing (SORT-Seq) is a popular procedure to select cells of interest for single-cell transcriptomics. However, FACS is not suitable for measurement of subcellular distribution of fluorescence or for small samples (<1,000 cells). The VYCAP puncher system overcomes these limitations. Here, we describe a workflow to capture, image, and collect fluorescent human retina pigment epithelium cells for SORT-Seq using this system. The workflow can be used for any cell type with a diameter of ∼5-50 μm. For complete details on the use and execution of this protocol, please refer to Segeren et al. (2020).

Keywords: Cell Biology; Genomics; Microscopy; Single Cell.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Base Sequence / genetics
Cell Separation / methods*
Exome Sequencing / methods
Flow Cytometry / methods
Gene Expression Profiling / methods
High-Throughput Nucleotide Sequencing / methods
Humans
Microscopy, Fluorescence / methods*
RNA / genetics
Sequence Analysis, RNA / methods
Single-Cell Analysis / methods*
Transcriptome / genetics
Workflow

Substances

RNA