신승원 (University of California Berkeley)

한빛사논문

조회198

University of California Berkeley

CV File 184 kb

Nat. Protoc., Jul 22 2024, | https://doi.org/10.1038/s41596-024-01021-y FIND-seq: high-throughput nucleic acid cytometry for rare single-cell transcriptomics

Seung Won Shin¹, Prakriti Mudvari², Shravan Thaploo³, Michael A. Wheeler³, Daniel C. Douek⁴, Francisco J. Quintana³, Eli A. Boritz², Adam R. Abate⁵ & Iain C. Clark¹

¹Department of Bioengineering, College of Engineering, California Institute for Quantitative Biosciences (QB3), University of California Berkeley, Berkeley, CA, USA.

²Virus Persistence and Dynamics Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.

³Ann Romney Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA.

⁴Human Immunology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.

⁵Department of Bioengineering and Therapeutic Sciences, School of Pharmacy, University of California San Francisco, San Francisco, CA, USA.

Corresponding author

Correspondence to Iain C. Clark.

Abstract

Rare cells have an important role in development and disease, and methods for isolating and studying cell subsets are therefore an essential part of biology research. Such methods traditionally rely on labeled antibodies targeted to cell surface proteins, but large public databases and sophisticated computational approaches increasingly define cell subsets on the basis of genomic, epigenomic and transcriptomic sequencing data. Methods for isolating cells on the basis of nucleic acid sequences powerfully complement these approaches by providing experimental access to cell subsets discovered in cell atlases, as well as those that cannot be otherwise isolated, including cells infected with pathogens, with specific DNA mutations or with unique transcriptional or splicing signatures. We recently developed a nucleic acid cytometry platform called 'focused interrogation of cells by nucleic acid detection and sequencing' (FIND-seq), capable of isolating rare cells on the basis of RNA or DNA markers, followed by bulk or single-cell transcriptomic analysis. This platform has previously been used to characterize the splicing-dependent activation of the transcription factor XBP1 in astrocytes and HIV persistence in memory CD4 T cells from people on long-term antiretroviral therapy. Here, we outline the molecular and microfluidic steps involved in performing FIND-seq, including protocol updates that allow detection and whole transcriptome sequencing of rare HIV-infected cells that harbor genetically intact virus genomes. FIND-seq requires knowledge of microfluidics, optics and molecular biology. We expect that FIND-seq, and this comprehensive protocol, will enable mechanistic studies of rare HIV⁺ cells, as well as other cell subsets that were previously difficult to recover and sequence.

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게재일2024년 07월 (BRIC 등록일 2024-07-24)
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