Abstract
Methods that measure the transcriptomic state of thousands of individual cells have transformed our understanding of cellular heterogeneity in eukaryotic cells since their introduction in the past decade. While simple and accessible protocols and commercial products are now available for the processing of mammalian cells, these existing technologies are incompatible with use in bacterial samples for several fundamental reasons including the absence of polyadenylation on bacterial messenger RNA, the instability of bacterial transcripts and the incompatibility of bacterial cell morphology with existing methodologies. Recently, we developed ProBac sequencing (ProBac-seq), a method that overcomes these technical difficulties and provides high-quality single-cell gene expression data from thousands of bacterial cells by using messenger RNA-specific probes. Here we provide details for designing large oligonucleotide probe sets for an organism of choice, amplifying probe sets to produce sufficient quantities for repeated experiments, adding unique molecular indexes and poly-A tails to produce finalized probes, in situ probe hybridization and single-cell encapsulation and library preparation. This protocol, from the probe amplification to the library preparation, requires ~7 d to complete. ProBac-seq offers several advantages over other methods by capturing only the desired target sequences and avoiding nondesired transcripts, such as highly abundant ribosomal RNA, thus enriching for signal that better informs on cellular state. The use of multiple probes per gene can detect meaningful single-cell signals from cells expressing transcripts to a lesser degree or those grown in minimal media and other environmentally relevant conditions in which cells are less active. ProBac-seq is also compatible with other organisms that can be profiled by in situ hybridization techniques.
Key points
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This protocol describes ProBac sequencing, a bacterial single-cell RNA sequencing method using droplet microfluidics and large oligonucleotide probe sets.
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ProBac sequencing offers advantages over other methods. By capturing only the desired target sequences and avoiding nondesired transcripts such as highly abundant ribosomal RNA, it better informs on cellular state. Using multiple probes per gene allows meaningful single-cell signals to be detected from less active cells or those expressing transcripts to a lesser degree.
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Data availability
The main data discussed in this protocol are available in the supporting primary research paper19. The raw datasets are available for research purposes upon reasonable request from the corresponding author.
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Acknowledgements
A.R. acknowledges funding from the University of North Carolina School of Medicine and the CGIBD institute at the University of North Carolina including by a grant from the National Institutes of Health, P30 DK034987. S.H. acknowledges funding from the National Institutes of Health National Institute of General Medical Sciences grant no. R00GM118910; National Institutes of Health National Heart, Lung, and Blood Institute grant no. R01HL158269; U19 Systems Immunology Pilot Project Grant at Harvard University and the Harvard University William F. Milton Fund. Portions of this research were conducted on the O2 High Performance Compute Cluster, supported by the Research Computing Group, at Harvard Medical School (see http://rc.hms.harvard.edu for more information).
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A.R. and P.S. conceived the manuscript. R.M., S.F.C., P.S. and A.R. performed the laboratory experiments. S.H. and R.M. created the data analysis pipeline. P.S. and A.R. wrote the paper with input from all other authors. A.R. and S.H. supervised the project.
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Nature Protocols thanks Kuanwei Sheng and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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McNulty, R. et al. Nat. Microbiol. 8, 934–945 (2023).
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Supplementary Information
Supplementary Figs. 1–6.
Supplementary Table 1
B. subtilis probes ordered from Twist Bioscience and shown here as a representation of an initial probe sequence
Supplementary Table 2
Primers for probe amplification and library generation. RCA primers shown here are specific for B. subtilis
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Samanta, P., Cooke, S.F., McNulty, R. et al. ProBac-seq, a bacterial single-cell RNA sequencing methodology using droplet microfluidics and large oligonucleotide probe sets. Nat Protoc (2024). https://doi.org/10.1038/s41596-024-01002-1
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DOI: https://doi.org/10.1038/s41596-024-01002-1
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