In our previous paper, Lubeck and Cai, we used super-resolution microscopy to resolve a large number of mRNAs in single cells. In this Correspondence, we present a sequential barcoding scheme to multiplex different mRNAs

Ahmad, Mubhij

Cai, Long

Coskun, Ahmet F.

Lubeck, Eric

Zhiyentayev, Timur

Caltech Authors - Main

Single cell in situ RNA profiling by sequential hybridization
Eric Lubeck1,2, Ahmet F. Coskun1,2, Timur Zhiyentayev1, Mubhij Ahmad1, and Long Cai1
1Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena,
CA, 91125
To the editor: In our previous paper, Lubeck and Cai1, we used super-resolution
microscopy to resolve a large number of mRNAs in single cells. In this correspondence, we
present a sequential barcoding scheme to multiplex different mRNAs.
Here, the mRNAs in cells are barcoded by sequential rounds of hybridization, imaging, and
probe stripping (Fig. 1a, Supplementary Fig. 1). As the transcripts are fixed in cells, the
corresponding fluorescent spots remain in place during multiple rounds of hybridization, and
can be aligned to read out a fluorophore sequence. This sequential barcode is designed to
uniquely identify an mRNA.
During each round of hybridization, we targeted each transcript by a set of FISH probes
labeled with a single type of fluorophore. We imaged the sample and then treated it with
DNAse I to remove the FISH probes. In a subsequent round the mRNA was hybridized with
the same FISH probes, but now labeled with a different dye. The number of barcodes
available scales as FN, where F is the number of fluorophores and N is the number of
hybridization rounds. For example, with 4 dyes, 8 rounds o=f hybridization can cover the
entire transcriptome (48=65,536).
As a demonstration, we barcoded 12 genes in single yeast cells with 4 dyes and 2 rounds of
hybridization (42=16, with 4 barcodes left out). Cells are immobilized on glass surfaces
(Supplementary Methods). The DNA probes are hybridized, imaged, and then removed by
DNAse I treatment (88.5±11.0% (SE) efficiency Supplementary Fig. 2). The remaining
signal is photobleached (Supplementary Fig. 3). Even after 6 hybridizations, mRNAs were
observed at 70.9±21.8% (SE) of the original intensity (Supplementary Fig. 4). We observed
that 77.9±5.6% (SE) of the spots that co-localized in the first two hybridizations also co-
localize with the third hybridization (Fig. 1b, Supplementary Figs. 5, 6). The mRNA
abundances are quantified by counting the occurrence of corresponding barcodes in the cell
(Supplementary Figs. 7, 8, n=37 cells). We also show that mRNAs can be stripped and re-
hybridized efficiently in adherent mammalian cells (Supplementary Figs. 9, 10).
Sequential barcoding has many advantages. First it scales up quickly; with even two dyes
the coding capacity is in principle unlimited. Second, during each hybridization, all available
Correspondence should be addressed to L.C. (lcai@caltech.edu).
2Equal Contribution
Competing financial interests
The authors declare no competing financial interests.
NIH Public Access
Author Manuscript
Nat Methods. Author manuscript; available in PMC 2014 July 08.
Published in final edited form as:
Nat Methods. 2014 April ; 11(4): 360–361. doi:10.1038/nmeth.2892.
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FISH probes against a transcript can be used, increasing the brightness of the FISH signal.
Lastly, barcode readout is robust, enabling full Z-stacks on native samples.
This barcoding scheme is conceptually akin to sequencing transcripts in single cells with
FISH. Compared to Ke et al2, our method takes advantage of the high hybridization
efficiency of FISH (>95% of the mRNAs are detected1,3) and the fact that base pair
resolution is usually not needed to uniquely identify a transcript. We note that FISH probes
can also be designed to resolve a large number of splice-isoforms, SNPs3, as well as
chromosome loci4 in single cells. In combination with our previous report of super-
resolution FISH1, the sequential barcoding method will enable the transcriptome to be
directly imaged at single cell resolution in complex samples, such as the brain.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
References
1. Lubeck E, Cai L. Nat. Methods. 2012; 9:743–748. [PubMed: 22660740]
2. Ke R, et al. Nat. Methods. 2013; 10:857–860. [PubMed: 23852452]
3. Levesque MJ, et al. Nat. Methods. 2013; 10:865–867. [PubMed: 23913259]
4. Levesque MJ, Raj A. Nat Meth. 2013; 10:246–248.
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Figure 1.
Sequential barcoding. (a) Schematic of sequential barcoding. In each round of hybridization,
24 probes are hybridized on each transcript, imaged and then stripped by DNAse I
treatment. The same probe sequences are used in different rounds of hybridization, but
probes are coupled to different fluorophores. (b) Composite four-color FISH Data from 3
rounds of hybridizations on multiple yeast cells. Twelve genes are encoded by 2 rounds of
hybridization, with the third hybridization using the same probes as hybridization 1. The
boxed regions are magnified in the bottom right corner of each image. The matching spots
are shown and barcodes are extracted. Spots without colocalization are due to nonspecific
binding of probes in the cell as well as mis-hybridization. The number of each barcode can
be quantified to provide the abundances of the corresponding transcripts in single cells.
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Single-cell in situ RNA profiling by sequential hybridization

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Single-cell in situ RNA profiling by sequential hybridization

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