Abstract
Sample multiplexing facilitates single‐cell sequencing by reducing costs, revealing subtle difference between similar samples, and identifying artifacts such as cell doublets. However, universal and cost‐effective strategies are rather limited. Here, we reported a concanavalin A‐based sample barcoding strategy (CASB), which could be followed by both single‐cell mRNA and ATAC (assay for transposase‐accessible chromatin) sequencing techniques. The method involves minimal sample processing, thereby preserving intact transcriptomic or epigenomic patterns. We demonstrated its high labeling efficiency, high accuracy in assigning cells/nuclei to samples regardless of cell type and genetic background, and high sensitivity in detecting doublets by three applications: 1) CASB followed by scRNA‐seq to track the transcriptomic dynamics of a cancer cell line perturbed by multiple drugs, which revealed compound‐specific heterogeneous response; 2) CASB together with both snATAC‐seq and scRNA‐seq to illustrate the IFN‐γ‐mediated dynamic changes on epigenome and transcriptome profile, which identified the transcription factor underlying heterogeneous IFN‐γ response; and 3) combinatorial indexing by CASB, which demonstrated its high scalability.
Highlights
Single-cell mRNA sequencing and single-nucleus assay for transposase-accessible chromatin using sequencing have emerged as powerful technologies for interrogating the heterogeneous transcriptional profiles and chromatin landscapes of multicellular subjects (Buenrostro, Wu et al, 2015, Cusanovich, Daza et al, 2015, Hashimshony, Wagner et al, 2012, Ramskold, Luo et al, 2012)
We developed a Concanavalin A-based Sample Barcoding strategy (CASB) that overcomes many of these limitations
The CASB consists of three components: biotinylated concanavalin A (ConA), streptavidin and biotinylated single-strand DNA (ssDNA) as barcoding molecules
Summary
Single-cell mRNA sequencing (scRNA-seq) and single-nucleus assay for transposase-accessible chromatin using sequencing (snATAC-seq) have emerged as powerful technologies for interrogating the heterogeneous transcriptional profiles and chromatin landscapes of multicellular subjects (Buenrostro, Wu et al, 2015, Cusanovich, Daza et al, 2015, Hashimshony, Wagner et al, 2012, Ramskold, Luo et al, 2012). With the ever-increasing throughput, these technologies have been used to reveal the temporal response of heterogeneous cell population under diverse perturbations, which require tens of samples to be processed in parallel (Hurley, Ding et al, 2020, Weinreb, Rodriguez-Fraticelli et al, 2020). Sample-specific barcodes (for example, Illumina library indices) are often incorporated at the very end of standard library preparation workflow. Such workflow requires parallel processing of multiple individual samples until the final step, is labor-intensive and limits the number of samples, and increase the reagent costs if a small number of cells would be sufficient to characterize the heterogeneity of each individual sample. Several methods have been developed in this endeavor, which introduce sample barcodes using either genetic or non-genetic mechanisms
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