High-throughput single-cell gene-expression profiling with multiplexed error-robust fluorescence in situ hybridization.
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 image-based 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 image-based 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.
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138; Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138; zhuang@chemistry.harvard.edu lmoffitt@mcb.harvard.edu.
Jeffrey R
JR
Moffitt
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138; Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138;
Junjie
J
Hao
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138; Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138;
Guiping
G
Wang
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138; Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138;
Kok Hao
KH
Chen
Center for Advanced Imaging, Harvard University, Cambridge, MA 02138;
Hazen P
0000-0003-4835-3692
HP
Babcock
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138; Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138; Center for Advanced Imaging, Harvard University, Cambridge, MA 02138; Department of Physics, Harvard University, Cambridge, MA 02138 zhuang@chemistry.harvard.edu lmoffitt@mcb.harvard.edu.
Xiaowei
0000-0002-6034-7853
X
Zhuang
D000465:Algorithms; D002455:Cell Division; D045744:Cell Line, Tumor; D004261:DNA Replication; D005456:Fluorescent Dyes / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D012313:RNA / Q000378:metabolism; D015203:Reproducibility of Results; D059010:Single-Cell Analysis / Q000379:methods*
D005456:Fluorescent Dyes; D012313:RNA
fluorescence; in situ hybridization; multiplexed imaging; single-cell analysis; transcriptomics