Spatially resolved, highly multiplexed RNA profiling in single cells

Multiplexed RNA imaging in single cells The basis of cellular function is where and when proteins are expressed and in what quantities. Single-molecule fluorescence in situ hybridization (smFISH) experiments quantify the copy number and location of mRNA molecules; however, the numbers of RNA species that can be simultaneously measured by smFISH has been limited. Using combinatorial labeling with error-robust encoding schemes, Chen et al. simultaneously imaged 100 to 1000 RNA species in a single cell. Such large-scale detection allows regulatory interactions to be analyzed at the transcriptome scale. Science, this issue p. 10.1126/science.aaa6090 A single-molecule imaging method allows simultaneous measurement of 1000 RNA species in single cells. INTRODUCTION The copy number and intracellular localization of RNA are important regulators of gene expression. Measurement of these properties at the transcriptome scale in single cells will give answers to many questions related to gene expression and regulation. Single-molecule RNA imaging approaches, such as single-molecule fluorescence in situ hybridization (smFISH), are powerful tools for counting and mapping RNA; however, the number of RNA species that can be simultaneously imaged in individual cells has been limited. This makes it challenging to perform transcriptomic analysis of single cells in a spatially resolved manner. Here, we report multiplexed error-robust FISH (MERFISH), a single-molecule imaging method that allows thousands of RNA species to be imaged in single cells by using combinatorial FISH labeling with encoding schemes capable of detecting and/or correcting errors. RATIONALE We labeled each cellular RNA with a set of encoding probes, which contain targeting sequences that bind the RNA and readout sequences that bind fluorescently labeled readout probes. Each RNA species is encoded with a particular combination of readout sequences. We used successive rounds of hybridization and imaging, each with a different readout probe, to identify the readout sequences bound to each RNA and to decode the RNA. In principle, combinatorial labeling allows the number of detectable RNA species to grow exponentially with the number of imaging rounds, but the detection errors also increase exponentially. To combat such accumulating errors, we exploited error-robust encoding schemes used in digital electronics, such as the extended Hamming code, in the design of our encoding probes but modified these schemes in order to account for the error properties in FISH measurements. We assigned each RNA a binary word in our modified Hamming code and encoded the RNA with a combination of readout sequences according to this binary word. RESULTS We first imaged 140 RNA species in human fibroblast cells using MERFISH with 16 rounds of hybridization and a modified Hamming code capable of both error detection and correction. We obtained ~80% detection efficiency and observed excellent correlation of RNA copy numbers determined with MERFISH with both bulk RNA sequencing data and conventional smFISH measurements of individual genes. Next, we used an alternative MERFISH encoding scheme, which is capable of detecting but not correcting errors, to image 1001 RNA species in individual cells using only 14 rounds of hybridization. The observed RNA copy numbers again correlate well with bulk sequencing data. However, the detection efficiency is only one-third that of the error-correcting encoding scheme. We performed correlation analysis of the 104 to 106 pairs of measured genes and identified many covarying gene groups that share common regulatory elements. Such grouping allowed us to hypothesize potential functions of ~100 unannotated or partially annotated genes of unknown functions. We further analyzed correlations in the spatial distributions of different RNA species and identified groups of RNAs with different distribution patterns in the cell. DISCUSSION This highly multiplexed imaging approach enables analyses based on the variation and correlation of copy numbers and spatial distributions of a large number of RNA species within single cells. Such analyses should facilitate the delineation of regulatory networks and in situ identification of cell types. We envision that this approach will allow spatially resolved transcriptomes to be determined for single cells. MERFISH for transcriptome imaging. Numerous RNA species can be identified, counted, and localized in a single cell by using MERFISH, a single-molecule imaging approach that uses combinatorial labeling and sequential imaging with encoding schemes capable of detection and/or correction of errors. This highly multiplexed measurement of individual RNAs can be used to compute the gene expression profile and noise, covariation in expression among different genes, and spatial distribution of RNAs within single cells. Knowledge of the expression profile and spatial landscape of the transcriptome in individual cells is essential for understanding the rich repertoire of cellular behaviors. Here, we report multiplexed error-robust fluorescence in situ hybridization (MERFISH), a single-molecule imaging approach that allows the copy numbers and spatial localizations of thousands of RNA species to be determined in single cells. Using error-robust encoding schemes to combat single-molecule labeling and detection errors, we demonstrated the imaging of 100 to 1000 distinct RNA species in hundreds of individual cells. Correlation analysis of the ~104 to 106 pairs of genes allowed us to constrain gene regulatory networks, predict novel functions for many unannotated genes, and identify distinct spatial distribution patterns of RNAs that correlate with properties of the encoded proteins.