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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.

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RNA biology is orchestrated by the dynamic interactions of RNAs and RNA‐binding proteins (RBPs). In the present study, we describe a new method of proximity‐dependent protein labeling to detect RNA–protein interactions [RNA‐bound protein proximity labeling (RBPL)]. We selected the well‐studied RNA‐binding protein PUF to examine the current proximity labeling enzymes birA* and APEX2. A new version of birA*, BASU, was used to validate that the PUF protein binds its RNA motif. We further optimized the RBPL labeling system using an inducible expression system. The RBPL (λN‐BASU) labeling experiments exhibited high signal‐to‐noise ratios. We subsequently determined that RBPL (λN‐BASU) is more suitable than RBPL (λN‐APEX2) for the detection of RNA–protein interactions in live cells. Interestingly, our results also reveal that proximity labeling is probably capable of biotinylating proximate nascent peptide.