Multimodal single-cell RNA sequencing enables the precise mapping of transcriptional and phenotypic features of cellular differentiation states but does not allow for simultaneous integration of critical posttranslational modification data. Researchers from the Peter MacCallum Cancer Centre describe SUrface-protein Glycan And RNA-seq (SUGAR-seq), a method that enables detection and analysis of N-linked glycosylation, extracellular epitopes, and the transcriptome at the single-cell level. Integrated SUGAR-seq and glycoproteome analysis identified tumor-infiltrating T cells with unique surface glycan properties that report their epigenetic and functional state.
Schematic representation of SUGAR-seq
TILs (CD3+ TCR+) were isolated from day 14 B16-Ova and MC38-Ova tumors (n = 6, pooled) by FACS. TILs were initially stained with biotinylated (1 μg/ml) and nonbiotinylated L-Pha (1:5 ratio), washed extensively, and then stained with ADT antibodies (anti–PD-1, anti-TIM3, and anti-biotin) and hash-tagging antibodies for sample demultiplexing. Single-cell capture was performed using the 10x Genomics platform.