High-throughput single-cell gene-expression profiling with multiplexed error-robust fluorescence in situ hybridization

Abstract

Image-based approaches to single-cell transcriptomics, in which RNA species are identified and counted in situ via imaging, have emerged as a powerful complement to single-cell methods based on RNA sequencing of dissociated cells. These image-based approaches naturally preserve the native spatial context of RNAs within a cell and the organization of cells within tissue, which are important for addressing many biological questions. However, the throughput of these imagebased approaches is relatively low. Here we report advances that lead to a drastic increase in the measurement throughput of multiplexed error-robust fluorescence in situ hybridization (MERFISH), an imagebased approach to single-cell transcriptomics. In MERFISH, RNAs are identified via a combinatorial labeling approach that encodes RNA species with error-robust barcodes followed by sequential rounds of single-molecule fluorescence in situ hybridization (smFISH) to read out these barcodes. Here we increase the throughput of MERFISH by two orders of magnitude through a combination of improvements, including using chemical cleavage instead of photobleaching to remove fluorescent signals between consecutive rounds of smFISH imaging, increasing the imaging field of view, and using multicolor imaging. With these improvements, we performed RNA profiling in more than 100,000 human cells, with as many as 40,000 cells measured in a single 18-h measurement. This throughput should substantially extend the range of biological questions that can be addressed by MERFISH.single-cell analysis | fluorescence | in situ hybridization | transcriptomics | multiplexed imaging

High-performance multiplexed fluorescence in situ hybridization in culture and tissue with matrix imprinting and clearing

“…Moreover, because these components are also a major source of autofluorescence, the autofluorescence background might be reduced by such an approach as well. To this end, we fixed the sample and hybridized it with oligonucleotide probes as in standard smFISH (1, 2) or MERFISH measurements (13,14), and then embedded the sample in an inert, nonfluorescence matrix to which RNA molecules were anchored, effectively imprinting the desired RNA signal onto this matrix (Fig. 1B).…”

“…Next, we performed labeling as in MERFISH experiments and tested whether off-target probe binding was indeed reduced by clearing. In a MERFISH measurement, we typically stain cells first with a complex library of "encoding" oligonucleotide probes (13,14). These encoding probes are not themselves fluorescently labeled.…”

“…S1 B and C). These barcodes are then measured in a series of hybridizations, each round of hybridization using either one fluorescently labeled "readout" probe complementary to a specific readout sequence, reading out one bit per round in the singlecolor imaging mode (13), or multiple readout probes labeled with spectrally distinct dyes, reading out multiple bits simultaneously in the multicolor imaging mode (14) (Fig. S1 B and C).…”

“…Most of these approaches take advantage of color multiplexing, which has allowed a few tens of RNA species to be imaged simultaneously. We have recently introduced multiplexed error-robust FISH (MERFISH), a massively multiplexed form of smFISH that allows RNA imaging and profiling at the transcriptomic scale (13,14). MERFISH achieves this level of multiplexing by assigning error-robust barcodes to individual RNA species, labeling RNAs combinatorically with oligonucleotide probes that contain a representation of these barcodes, and reading out these barcodes through sequential rounds of single-color (13) or multicolor (14) smFISH imaging (Fig.…”

“…Using this approach, we have imaged 140 and 1,000 RNA species in individual cells with two different encoding schemes, one of which allows both error detection and correction and the other that allows error detection (13), and the number of addressable RNA species can be further changed by using different encoding schemes. Recently, we have increased the measurement throughput of MERFISH and demonstrated the ability to profile gene expression in tens of thousands of cells in a single day-long measurement (14). A different multiplexed smFISH method using color-based barcodes and sequential imaging (seqFISH) has been independently proposed and initially demonstrated with measurements of 12 RNA species in individual cells (12).…”