Microfluidics-free single-cell genomics with templated emulsification

Single-cell RNA sequencing is now a standard method used to reveal the molecular details of cellular heterogeneity, but current approaches have limitations on speed, scale, and ease of use that stem from the complex microfluidic devices or fluid handling steps required for sample processing. We, the...

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Bibliographic Details
Published in:bioRxiv 2022-10
Main Authors: Clark, Iain C, Fontanez, Kristina M, Meltzer, Robert H, Xue, Yi, Hayford, Corey, May-Zhang, Aaron, D'amato, Chris, Osman, Ahmad, Zhang, Jesse Q, Hettige, Pabodha, Jacob Sa Ishibashi, Delley, Cyrille L, Weisgerber, Daniel W, Replogle, Joseph M, Jost, Marco, Phong, Kiet T, Kennedy, Vanessa E, Cheryl Ac Peretz, Kim, Esther A, Song, Siyou, Karlon, William, Weissman, Jonathan S, Smith, Catherine C, Gartner, Zev J, Abate, Adam
Format: Article
Language:English
Subjects:
Breast
Chemotherapy
Emulsification
Lysis
Microfluidics
Phenotypes
Transcriptomes
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Summary:Single-cell RNA sequencing is now a standard method used to reveal the molecular details of cellular heterogeneity, but current approaches have limitations on speed, scale, and ease of use that stem from the complex microfluidic devices or fluid handling steps required for sample processing. We, therefore, developed a method that does not require specialized microfluidic devices, expertise, or hardware. Our approach is based on particle-templated emulsification, which allows single-cell encapsulation and barcoding of cDNA in uniform droplet emulsions with only a vortexer. PIP-seq accommodates a wide range of emulsification formats, including microwell plates and large-volume conical tubes, enabling thousands of samples or millions of cells to be processed in minutes. We demonstrate that PIP-seq produces high-purity transcriptomes in mouse-human mixing studies, is compatible with multi-omics measurements, and can accurately characterize cell types in human breast tissue when compared to a commercial microfluidic platform. Single-cell transcriptional profiling of mixed phenotype acute leukemia using PIP-seq revealed the emergence of heterogeneity within chemotherapy-resistant cell subsets that were hidden by standard immunophenotyping. PIP-seq is a simple, flexible, and scalable next-generation workflow that extends single-cell sequencing to new applications, including screening, diagnostics, and disease monitoring. Competing Interest Statement ARA filed a patent related to PIPs and is a founder of Fluent Biosciences. ICC consults for Fluent Biosciences and is on their Scientific Advisory Board. KMF, RM, YX, CH, AO, PH, JQZ, AM-Z, and CDA are employees at Fluent Biosciences and are working to commercialize the PIP-seq technology. MJ consults for Maze Therapeutics and Gate Biosciences. JMR consults for Maze Therapeutics and Waypoint Bio. JSW declares outside interest in 5 AM Venture, Amgen, Chroma Medicine, DEM Biosciences, KSQ Therapeutics, Maze Therapeutics, Tenaya Therapeutics, Tessera Therapeutics and Velia Therapeutics. Footnotes * New data includes: 1. A comparison between PIP-seq to other technologies across a range of sequencing depths. 2. PIP-seq on 138,000 breast tissue cells using a single-tube emulsification. 3. The re-sequencing of breast tissue samples at significantly higher depth (36,5000 reads per cell). 4. Antibody-tag-based cell hashing 5. Additional plate-based emulsification experiments in 96- and 384-well formats show PK-based cell lysis
DOI:10.1101/2022.06.10.495582