Isolation and RNA sequencing of single nuclei from Drosophila tissues

Colleen N McLaughlin; Yanyan Qi; Stephen R Quake; Liqun Luo; Hongjie Li

doi:10.1016/j.xpro.2022.101417

Isolation and RNA sequencing of single nuclei from Drosophila tissues

STAR Protoc. 2022 May 20;3(2):101417. doi: 10.1016/j.xpro.2022.101417. eCollection 2022 Jun 17.

Authors

Colleen N McLaughlin¹, Yanyan Qi^{2

3}, Stephen R Quake^{4

5}, Liqun Luo¹, Hongjie Li^{2

3}

Affiliations

¹ Department of Biology, Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305, USA.
² Huffington Center on Aging, Baylor College of Medicine, Houston, TX 77030, USA.
³ Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.
⁴ Department of Bioengineering and Applied Physics, Stanford University, Stanford, CA, USA.
⁵ Chan Zuckerberg Biohub, San Francisco, CA, USA.

Abstract

Many insect cells are encapsulated within the exoskeleton and cannot be dissociated intact, making them inaccessible to single-cell transcriptomic profiling. We have used single-nucleus RNA sequencing to extract transcriptomic information from multiple Drosophila tissues. Here, we describe procedures for the (1) dissociation of single nuclei, (2) isolation of single nuclei using two popular cell sorters, and (3) preparation of libraries for Smart-seq2 and 10× Genomics. This protocol enables generation of high-quality transcriptomes from single nuclei and can be applied to other species. For complete details on the use and execution of this protocol, please refer to McLaughlin et al. (2021) and Li et al. (2022).

Keywords: Genomics; Model Organisms; RNAseq; Sequencing.

Publication types

Research Support, Non-U.S. Gov't
Research Support, N.I.H., Extramural

MeSH terms

Animals
Base Sequence
Drosophila* / genetics
Exome Sequencing
Gene Expression Profiling* / methods
Sequence Analysis, RNA / methods

Abstract

Publication types

MeSH terms

Grants and funding