Overloading And unpacKing (OAK) - droplet-based combinatorial indexing for ultra-high throughput single-cell multiomic profiling

• Published in: Nature communications 2024-10, Vol.15 (1), p.9146-12, Article 9146
• Main Authors: Wu, Bing, Bennett, Hayley M., Ye, Xin, Sridhar, Akshayalakshmi, Eidenschenk, Celine, Everett, Christine, Nazarova, Evgeniya V., Chen, Hsu-Hsin, Kim, Ivana K., Deangelis, Margaret, Owen, Leah A., Chen, Cynthia, Lau, Julia, Shi, Minyi, Lund, Jessica M., Xavier-Magalhães, Ana, Patel, Neha, Liang, Yuxin, Modrusan, Zora, Darmanis, Spyros
• Format: Article
• Language: English
• Subjects: 38/23; 38/39; 38/91; 631/1647/514/1949; 631/337/2019; 631/45/612/100; 631/61/350/877; Bar codes; Cell differentiation; Cell Line, Tumor; Chromatin; Combinatorial analysis; Complexity; Droplets; Epithelial cells; Epithelium; Gene Expression Profiling - methods; Gene sequencing; High-Throughput Nucleotide Sequencing - methods; Humanities and Social Sciences; Humans; Indexing; Melanoma; Melanoma - drug therapy; Melanoma - genetics; Melanoma - pathology; Microfluidics; multidisciplinary; Overloading; Reagents; Science; Science (multidisciplinary); Sensitivity analysis; Sequence Analysis, RNA - methods; Single-Cell Analysis - methods; Transcriptome; Transcriptomics
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-024-53227-z

Multiomic profiling of single cells by sequencing is a powerful technique for investigating cellular diversity. Existing droplet-based microfluidic methods produce many cell-free droplets, underutilizing bead barcodes and reagents. Combinatorial indexing on microplates is more efficient for barcoding but labor-intensive. Here we present Overloading And unpacKing (OAK), which uses a droplet-based barcoding system for initial compartmentalization followed by a second aliquoting round to achieve combinatorial indexing. We demonstrate OAK’s versatility with single-cell RNA sequencing as well as paired single-nucleus RNA sequencing and accessible chromatin profiling. We further showcase OAK’s performance on complex samples, including differentiated bronchial epithelial cells and primary retinal tissue. Finally, we examine transcriptomic responses of over 400,000 melanoma cells to a RAF inhibitor, belvarafenib, discovering a rare resistant cell population (0.12%). OAK’s ultra-high throughput, broad compatibility, high sensitivity, and simplified procedures make it a powerful tool for large-scale molecular analysis, even for rare cells. Single-cell sequencing approaches need to balance sensitivity, throughput and experimental complexity. Here the authors combine droplet-based microfluidics and combinatorial indexing to develop OAK, a versatile method for ultra-high throughput single-cell multiomic profiling.