An Advanced Single-Cell RNA Sequencing (scRNA-seq) Protocol Utilizing Custom-Designed Multiplexing.

While cell hashing enhances single-cell RNA sequencing (scRNA-seq) efficiency and minimizes batch effects, commercial mouse hashtags often fail in FVB/N and several other strains due to antibody-epitope incompatibility. We describe a robust alternative utilizing biotinylated antibody cocktails and streptavidin-conjugated oligos to enable reliable sample multiplexing. This approach was validated in FVB/N lung tissues, yielding high-quality single-cell libraries. Our protocol offers a practical solution for researchers requiring strain-specific or custom-designed multiplexing strategies for single-cell transcriptomics. Key features • Strain-specific compatibility: Resolves the known H-2q haplotype mismatch in FVB/N mice that fails standard commercial MHC-I hashtag antibodies in cell hashing. • Multi-omic 5' workflow integration: Enables simultaneous sample multiplexing with 10× Genomics 5 chemistry, facilitating joint gene expression and V(D)J repertoire (TCR/BCR) profiling. • Enhanced non-immune cell labeling: Incorporates anti-CD326 (Ep-CAM) to ensure robust hashing of epithelial and tumor cells that may exhibit MHC-I downregulation or lack CD45. • Customizable biotin-streptavidin framework: Provides a modular system using biotinylated antibody cocktails and streptavidin-barcodes, adaptable for any mouse strain or tissue-specific cell markers.