MULTI-seq: sample multiplexing for single-cell RNA sequencing using lipid-tagged indices

• Published in: Nature methods Vol. 16; no. 7; pp. 619 - 626
• Main Authors: McGinnis, Christopher S., Patterson, David M., Winkler, Juliane, Conrad, Daniel N., Hein, Marco Y., Srivastava, Vasudha, Hu, Jennifer L., Murrow, Lyndsay M., Weissman, Jonathan S., Werb, Zena, Chow, Eric D., Gartner, Zev J.
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.07.2019; Nature Publishing Group
• Subjects: 631/1647/2017; 631/208/199; 631/337/2019; Analysis; Animals; Artifact identification; Base Sequence; Bioinformatics; Biological Microscopy; Biological Techniques; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Breast cancer; Cancer; Cell activation; Cell viability; Cryopreservation; Epithelial cells; Gene expression; Gene sequencing; Genetic aspects; Health aspects; HEK293 Cells; High-Throughput Nucleotide Sequencing; Humans; Life Sciences; Lipids; Lipids - chemistry; Lymphocytes T; Mammary gland; Metastases; Methods; Molecular dynamics; Multiplexing; Nuclei (cytology); Oncology, Experimental; Perturbation; Proteomics; Reagents; Ribonucleic acid; RNA; RNA sequencing; Sequence Analysis, RNA - methods; Single-Cell Analysis - methods; Tagging; Xenografts; Xenotransplantation; United States
• ISSN: 1548-7091; 1548-7105; 1548-7105
• DOI: 10.1038/s41592-019-0433-8

Sample multiplexing facilitates scRNA-seq by reducing costs and identifying artifacts such as cell doublets. However, universal and scalable sample barcoding strategies have not been described. We therefore developed MULTI-seq: multiplexing using lipid-tagged indices for single-cell and single-nucleus RNA sequencing. MULTI-seq reagents can barcode any cell type or nucleus from any species with an accessible plasma membrane. The method involves minimal sample processing, thereby preserving cell viability and endogenous gene expression patterns. When cells are classified into sample groups using MULTI-seq barcode abundances, data quality is improved through doublet identification and recovery of cells with low RNA content that would otherwise be discarded by standard quality-control workflows. We use MULTI-seq to track the dynamics of T-cell activation, perform a 96-plex perturbation experiment with primary human mammary epithelial cells and multiplex cryopreserved tumors and metastatic sites isolated from a patient-derived xenograft mouse model of triple-negative breast cancer. Tagging live single cells and nuclei with lipid- or cholesterol-modified oligonucleotides enables massive scRNA-seq sample multiplexing, identifies doublets and recovers cells with low RNA content.