Highly specific multiplexed RNA imaging in tissues with split-FISH.

We present split-FISH, a multiplexed fluorescence in situ hybridization method that leverages a split-probe design to achieve enhanced specificity. Split-FISH reduces off-target background fluorescence, decreases false positives and enables accurate RNA profiling in uncleared tissues. We demonstrate the efficacy of split-FISH on various mouse tissues by quantifying the distribution and abundance of 317 genes in single cells and reveal diverse localization patterns for spatial regulation of the transcriptome in complex tissues.

Author Correction: Highly specific multiplexed RNA imaging in tissues with split-FISH.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Highly sensitive spatial transcriptomics using FISHnCHIPs of multiple co-expressed genes.

High-dimensional, spatially resolved analysis of intact tissue samples promises to transform biomedical research and diagnostics, but existing spatial omics technologies are costly and labor-intensive. We present Fluorescence In Situ Hybridization of Cellular HeterogeneIty and gene expression Programs (FISHnCHIPs) for highly sensitive in situ profiling of cell types and gene expression programs. FISHnCHIPs achieves this by simultaneously imaging ~2-35 co-expressed genes (clustered into modules) that are spatially co-localized in tissues, resulting in similar spatial information as single-gene Fluorescence In Situ Hybridization (FISH), but with ~2-20-fold higher sensitivity. Using FISHnCHIPs, we image up to 53 modules from the mouse kidney and mouse brain, and demonstrate high-speed, large field-of-view profiling of a whole tissue section. FISHnCHIPs also reveals spatially restricted localizations of cancer-associated fibroblasts in a human colorectal cancer biopsy. Overall, FISHnCHIPs enables fast, robust, and scalable cell typing of tissues with normal physiology or undergoing pathogenesis.

Author Correction: Reference component analysis of single-cell transcriptomes elucidates cellular heterogeneity in human colorectal tumors.

In the version of the article published, the author list is not accurate. Igor Cima and Min-Han Tan should have been authors, appearing after Mark Wong in the author list, while Paul Jongjoon Choi should not have been listed as an author. Igor Cima and Min-Han Tan both have the affiliation Institute of Bioengineering and Nanotechnology, Singapore, Singapore, and their contributions should have been noted in the Author Contributions section as "I.C. preprocessed Primary Cell Atlas data with inputs from M.-H.T." The following description of the contribution of Paul Jongjoon Choi should not have appeared: "P.J.C. supported the smFISH experiments." In the 'RCA: global panel' section of the Online Methods, the following sentence should have appeared as the second sentence, "An expression atlas of human primary cells (the Primary Cell Atlas) was preprocessed similarly to in ref. 55," with new reference 55 (Cima, I. et al. Tumor-derived circulating endothelial cell clusters in colorectal cancer. Science Transl. Med. 8, 345ra89, 2016).

Reference component analysis of single-cell transcriptomes elucidates cellular heterogeneity in human colorectal tumors.

Intratumoral heterogeneity is a major obstacle to cancer treatment and a significant confounding factor in bulk-tumor profiling. We performed an unbiased analysis of transcriptional heterogeneity in colorectal tumors and their microenvironments using single-cell RNA-seq from 11 primary colorectal tumors and matched normal mucosa. To robustly cluster single-cell transcriptomes, we developed reference component analysis (RCA), an algorithm that substantially improves clustering accuracy. Using RCA, we identified two distinct subtypes of cancer-associated fibroblasts (CAFs). Additionally, epithelial-mesenchymal transition (EMT)-related genes were found to be upregulated only in the CAF subpopulation of tumor samples. Notably, colorectal tumors previously assigned to a single subtype on the basis of bulk transcriptomics could be divided into subgroups with divergent survival probability by using single-cell signatures, thus underscoring the prognostic value of our approach. Overall, our results demonstrate that unbiased single-cell RNA-seq profiling of tumor and matched normal samples provides a unique opportunity to characterize aberrant cell states within a tumor.