Core Leader: Peter Sims

The Advanced Single Cell Technology Innovation Core (ASCeTIC) will provide state-of-the-art technology for single-cell genomic analysis, multiplexed single-cell molecular perturbation, and in situ validation, leveraging robust experimental pipelines for large-scale profiling of human and murine solid tumors. A key Center deliverable is the identification of Master Regulator (MR) proteins underlying drug response and cell-cell interactions in the tumor microenvironment (TME), by interrogating tumor-specific regulatory networks. ASCeTIC will combine scRNA-seq with multiplexed CRISPR perturbations of putative MRs for direct validation of their downstream targets. To this end, we have successfully implemented CRISPR droplet sequencing (CROP-seq) for multiplexed, pooled gene perturbation with direct read-out by scRNA-seq (1). The Core will generate pooled lentiviral gRNA constructs for multiplexed perturbation and validation of master regulators with CROP-seq.

The Core will assist CaST investigators in their efforts to deconvolve cell type-specific drug responses in cultured slices of solid tumors using scRNA-seq. A major challenge is distinguishing cell death from reprogramming as the drug-induced loss of cells could occur via either mechanism. Furthermore, programmed cell death (e.g. apoptosis) generally occurs post-transcriptionally and may not induce a specific gene expression signature. The Core will provide SCOPE-seq, a highly scalable technology (2) for directly linking of live cell imaging or cytometry with scRNA-seq. SCOPE-seq combines fluorescent indicators of apoptosis and cell viability with scRNA-seq to unambiguously identify drug responsive subpopulations. Additionally, the Core will provide a suite of imaging- and spatial analysis tools for targeted in situ validation of findings from single-cell genomics and analysis of cell-cell interactions, including multiplexed RNA-FISH using RNAScope technology and immunohistochemical (IHC) analysis at the protein level. Our goals include:

Providing end-to-end experimental pipelines for single-cell RNA-seq (scRNA-seq), joint analysis of gene expression and chromatin state (scRNA/ATAC-seq), and joint analysis of cellular imaging and gene expression (SCOPE-seq). ASCeTIC will provide robust protocols and platforms for sample preparation, quality control, single-cell capture, single-cell genomic library construction, and sequencing to Center investigators using commercial technology from 10x Genomics and Illumina. We will also offer SCOPE-seq, a new microfluidic technology for linking live cell imaging or cytometry with scRNA-seq at scale.

Producing pooled lentiviral libraries for joint CRISPR perturbation and scRNA-seq readout. The Core will generate gRNA libraries for CROP-seq analysis of the molecular effect of candidate MR silencing (3) and will provide commensurate single-cell analyses.

Providing access to imaging-based validation tools and multiplexed spatial proteomics technologies. For targeted in situ validation of key findings from single-cell genomic analysis, ASCeTIC will provide RNA- and protein-level analysis using RNAScope and IHC, respectively. We will also offer both cyclic immunofluorescence for spatially resolved, highly multiplexed proteomic analysis.


1. Datlinger P, Rendeiro AF, Schmidl C, Krausgruber T, Traxler P, Klughammer J, Schuster LC, Kuchler A, Alpar D, Bock C. Pooled CRISPR screening with single-cell transcriptome readout. Nat Methods. 2017;14(3):297-301. Epub 20170118. doi: 10.1038/nmeth.4177. PubMed PMID: 28099430; PMCID: PMC5334791.

2. Yuan J, Sheng J, Sims PA. SCOPE-Seq: a scalable technology for linking live cell imaging and single-cell RNA sequencing. Genome Biol. 2018;19(1):227. Epub 20181224. doi: 10.1186/s13059-018-1607-x. PubMed PMID: 30583733; PMCID: PMC6305572.

3. Kong W, Biddy BA, Kamimoto K, Amrute JM, Butka EG, Morris SA. CellTagging: combinatorial indexing to simultaneously map lineage and identity at single-cell resolution. Nat Protoc. 2020;15(3):750-72. Epub 20200212. doi: 10.1038/s41596-019-0247-2. PubMed PMID: 32051617; PMCID: PMC7427510.