Photo-sensitive oligonucleotides combined with topologically imposed light gradients enable spatially-resolved single-cell transcriptomics and epigenomics

The organization of cells within a tissue plays a critical role in tuning cellular function. Several methods have been recently developed to capture the transcriptome of single cells while retaining positional information. However, these genome-wide sequencing methods typically do not have the spatial resolution of individual cells and are limited to quantifying positional information within an arranged lattice, thereby failing to capture regions of nonoverlapping tissue. Further, these methods are generally limited to profiling fixed cells with reduced mRNA capture efficiency compared to standard scRNA-seq. In addition, existing methods lack modularity and cross-platform compatibility, thereby limiting most of these techniques from jointly profiling the genome-wide epigenetic state of cells. To overcome these limitations, we present scSTAMP-seq (single-cell Spatial Transcriptomic And Multiomic Profiling), an approach that employs cholesterol-tagged photolabile oligonucleotides that incorporate into cell membranes, enabling us to “stamp” the position of each cell using patterned light prior to tissue dissociation and single-cell sequencing. Applied to live cells, scSTAMP-seq efficiently captures spatially resolved single-cell transcriptomes and at a high resolution for all cells within a field of view. Furthermore, we showcase the dynamic spatial resolution provided by light gradients, enabling us to accurately map the position of individual cells. Finally, we show that scSTAMP-seq is modular and can be seamlessly integrated with various downstream single-cell sequencing technologies. We demonstrate this by performing scRNA-seq using plate- and droplet-based methods, and by performing joint epigenome and transcriptome sequencing from the same cell while preserving positional information. Collectively, these results demonstrate that scSTAMP-seq is a sensitive and high-throughput technology for mapping single-cell transcriptomes and epigenomes at the spatial resolution of individual cells. Overall design: scSTAMP-seq profiles live Hela and U2OS cell cultures throughout the study. Live cell samples were incubated with photocleavable hashtag oligos (PHOs) that embed into the cell membrane through cholesterol modifications to oligonucleotide anchors that hybridize with PHOs, enabling one to "stamp" the position of each cell using spatially imposed light gradients prior to tissue dissociation and single-cell sequencing. By quantifying the extent of PHO cleavage that is proportional to light exposure of each cell, together with mRNA from the same cell, enables single-cell transcriptomics that retains positional information of cells within a sample. For precise spatial light patterning (~500 nm), a digital micromirror device (DMD) was used to deliver UV light to illuminate cell samples. For greater illumination area of cell samples, a UV LED floodlight was implemented.