ProBac-seq, a bacterial single-cell RNA sequencing methodology using droplet microfluidics and large oligonucleotide probe sets

Prosenjit Samanta, Samuel F. Cooke, Ryan McNulty, Sahand Hormoz, Adam Rosenthal

Published: 2024-05-19 DOI: 10.1038/s41596-024-01002-1

cellular heterogeneity

bacterial single-cell RNA sequencing

ProBac-seq

oligonucleotide probe sets

droplet microfluidics

AI 解读

Supplementary information

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

Efficient and safe single-cell cloning of human pluripotent stem cells using the CEPT cocktail

Profiling native pulmonary basement membrane stiffness using atomic force microscopy

Genetic circuit design automation with Cello 2.0

Synthesis of siRNA nanoparticles to silence plaque-destabilizing gene in atherosclerotic lesional macrophages

Scalable dual-omics profiling with single-nucleus chromatin accessibility and mRNA expression sequencing 2 (SNARE-seq2)

A practical guide to scanning light-field microscopy with digital adaptive optics

Effective high-throughput isolation of fully human antibodies targeting infectious pathogens

Improved sampling and DNA extraction procedures for microbiome analysis in food-processing environments

MOF-derived nanoporous carbons with diverse tunable nanoarchitectures

Tutorial: a statistical genetics guide to identifying HLA alleles driving complex disease

查看全部

Sections

Figures

References

Supplementary information

Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl Acad. Sci. USA 102, 15545–15550 (2005).
Tang, F., Lao, K. & Surani, M. A. Development and applications of single-cell transcriptome analysis. Nat. Methods 8, S6–S11 (2011).
Ozsolak, F. & Milos, P. M. RNA sequencing: advances, challenges and opportunities. Nat. Rev. Genet. 12, 87–98 (2011).
Fan, H. C., Fu, G. K. & Fodor, S. P. A. Expression profiling. Combinatorial labeling of single cells for gene expression cytometry. Science 347, 1258367 (2015).
Klein, A. M. et al. Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells. Cell 161, 1187–1201 (2015).
Macosko, E. Z. et al. Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. Cell 161, 1202–1214 (2015).
Cao, J. et al. Comprehensive single-cell transcriptional profiling of a multicellular organism. Science 357, 661–667 (2017).
Zheng, G. X. Y. et al. Massively parallel digital transcriptional profiling of single cells. Nat. Commun. 8, 14049 (2017).
Rosenberg, A. B. et al. Single-cell profiling of the developing mouse brain and spinal cord with split-pool barcoding. Science 360, 176–182 (2018).
Rodriques, S. G. et al. Slide-seq: a scalable technology for measuring genome-wide expression at high spatial resolution. Science 363, 1463–1467 (2019).
Hashimshony, T., Wagner, F., Sher, N. & Yanai, I. CEL-seq: single-cell RNA-seq by multiplexed linear amplification. Cell Rep. 2, 666–673 (2012).
Jovic, D. et al. Single-cell RNA sequencing technologies and applications: a brief overview. Clin. Transl. Med. 12, e694 (2022).
Sheng, K., Cao, W., Niu, Y., Deng, Q. & Zong, C. Effective detection of variation in single-cell transcriptomes using MATQ-seq. Nat. Methods 14, 267–270 (2017).
Imdahl, F., Vafadarnejad, E., Homberger, C., Saliba, A. E. & Vogel, J. Single-cell RNA-sequencing reports growth-condition-specific global transcriptomes of individual bacteria. Nat. Microbiol. 5, 1202–1206 (2020).
Blattman, S. B., Jiang, W., Oikonomou, P. & Tavazoie, S. Prokaryotic single-cell RNA sequencing by in situ combinatorial indexing. Nat. Microbiol. 5, 1192–1201 (2020).
Kuchina, A. et al. Microbial single-cell RNA sequencing by split-pool barcoding. Science 371, eaba5257 (2021).
Wang, B. et al. Single-cell massively-parallel multiplexed microbial sequencing (M3-seq) identifies rare bacterial populations and profiles phage infection. Nat. Microbiol. http://doi.org/10.1038/s41564-023-01462-3 (2023).
Ma, P. et al. Bacterial droplet-based single-cell RNA-seq reveals antibiotic-associated heterogeneous cellular states. Cell 186, 877–891.e14 (2023).
McNulty, R. et al. Probe-based bacterial single-cell RNA sequencing predicts toxin regulation. Nat. Microbiol. 8, 934–945 (2023).
Xu, Z. et al. Droplet-based high-throughput single microbe RNA sequencing by smRandom-seq. Nat. Commun. 14, 5130 (2023).
Phillips, R. M. Cell Biology by the Numbers (Garland Science, 2015).
Régnier, P. & Marujo, P. E. Polyadenylation and degradation of RNA in Prokaryotes. In Madame Curie Bioscience Database (Landes Bioscience, 2013).
Dar, D., Dar, N., Cai, L. & Newman, D. K. Spatial transcriptomics of planktonic and sessile bacterial populations at single-cell resolution. Science 373, eabi4882 (2021).
Raj, A. Single-molecule RNA FISH. in Encyclopedia of Biophysics (ed. Roberts, G. C. K.) 2340–2343 (Springer, 2013).
Taniguchi, Y. et al. Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single cells. Science 329, 533–538 (2010).
Jia, Z., Dong, Y., Xu, H. & Wang, F. Optimizing the hybridization chain reaction–fluorescence in situ hybridization (HCR–FISH) protocol for detection of microbes in sediments. Mar. Life Sci. Technol. 3, 529–541 (2021).
Pavlekovic, M. et al. Optimization of three FISH procedures for in situ detection of anaerobic ammonium oxidizing bacteria in biological wastewater treatment. J. Microbiol. Methods 78, 119–126 (2009).
Glöckner, F. O. et al. An in situ hybridization protocol for detection and identification of planktonic bacteria. Syst. Appl. Microbiol. 19, 403–406 (1996).
Potapov, V. & Ong, J. L. Examining sources of error in PCR by single-molecule sequencing. PloS ONE 12, e0169774 (2017).
Dragosits, M. & Mattanovich, D. Adaptive laboratory evolution—principles and applications for biotechnology. Microb. Cell Fact. 12, 64 (2013).
Lenski, R. E. Experimental evolution and the dynamics of adaptation and genome evolution in microbial populations. ISME J. 11, 2181–2194 (2017).
Bastian, F. et al. Eukaryotic cell capture by amplified magnetic in situ hybridization using yeast as a model. Front. Microbiol. 12, 759478 (2021).
Chen, S.-H., Lo, C.-Z., Tsai, M.-C., Hsiung, C. A. & Lin, C.-Y. The unique probe selector: a comprehensive web service for probe design and oligonucleotide arrays. BMC Bioinforma. 9, S8 (2008).
Schmidt, T. L. et al. Scalable amplification of strand subsets from chip-synthesized oligonucleotide libraries. Nat. Commun. 6, 8634 (2015).
Rosenthal, A. Z. et al. Metabolic interactions between dynamic bacterial subpopulations. eLife 7, e33099 (2018).
Tsoi, R. et al. Metabolic division of labor in microbial systems. Proc. Natl Acad. Sci. USA 115, 2526–2531 (2018).
Mridha, S. & Kümmerli, R. Coordination of siderophore gene expression among clonal cells of the bacterium Pseudomonas aeruginosa. Commun. Biol. 5, 1–12 (2022).
Armbruster, C. R. et al. Heterogeneity in surface sensing suggests a division of labor in Pseudomonas aeruginosa populations. eLife 8, e45084 (2019).
Akerley, B. J., Cotter, P. A. & Miller, J. F. Ectopic expression of the flagellar regulon alters development of the bordetella–host interaction. Cell 80, 611–620 (1995).
Lyu, Z., Yang, A., Villanueva, P., Singh, A. & Ling, J. Heterogeneous flagellar expression in single salmonella cells promotes diversity in antibiotic tolerance. mBio 12, e0237421 (2021).
Zuo, W. & Wu, Y. Dynamic motility selection drives population segregation in a bacterial swarm. Proc. Natl Acad. Sci. USA 117, 4693–4700 (2020).
Garrett, E. M. et al. Phase variation of a signal transduction system controls Clostridioides difficile colony morphology, motility, and virulence. PLoS Biol. 17, e3000379 (2019).
Adiciptaningrum, A. M., Blomfield, I. C. & Tans, S. J. Direct observation of type 1 fimbrial switching. EMBO Rep. 10, 527–532 (2009).
Yu, Q. et al. The Agr-like quorum sensing system is required for pathogenesis of necrotic enteritis caused by Clostridium perfringens in poultry. Infect. Immun. 85, e00975–16 (2017).
Arnoldini, M. et al. Bistable expression of virulence genes in salmonella leads to the formation of an antibiotic-tolerant subpopulation. PLoS Biol. 12, e1001928 (2014).
Hockenberry, A. M. et al. Microbiota-derived metabolites inhibit Salmonella virulent subpopulation development by acting on single-cell behaviors. Proc. Natl Acad. Sci. USA 118, e2103027118 (2021).
Penaranda, C. & Hung, D. T. Single-cell RNA sequencing to understand host–pathogen interactions. ACS Infect. Dis. 5, 336–344 (2019).
Fuchs, B. M. et al. Flow cytometric analysis of the in situ accessibility of Escherichia coli 16S rRNA for fluorescently labeled oligonucleotide probes. Appl. Environ. Microbiol. 64, 4973–4982 (1998).
Singh, L. & Jones, K. W. The use of heparin as a simple cost-effective means of controlling background in nucleic acid hybridization procedures. Nucleic Acids Res. 12, 5627–5638 (1984).
Takei, N. et al. High-sensitivity and high-resolution in situ hybridization of coding and long non-coding RNAs in vertebrate ovaries and testes. Biol. Proced. Online 20, 6 (2018).
Hutton, J. R. Renaturation kinetics and thermal stability of DNA in aqueous solutions of formamide and urea. Nucleic Acids Res. 4, 3537–3555 (1977).
Beliveau, B. J. et al. OligoMiner provides a rapid, flexible environment for the design of genome-scale oligonucleotide in situ hybridization probes. Proc. Natl Acad. Sci. USA 115, E2183–E2192 (2018).
Rouillard, J.-M., Zuker, M. & Gulari, E. OligoArray 2.0: design of oligonucleotide probes for DNA microarrays using a thermodynamic approach. Nucleic Acids Res. 31, 3057–3062 (2003).
Srivastava, G. P. & Xu, D. Genome-scale PCR primer design. In PCR Primer Design (ed. Yuryev, A.) 159–175 (Humana Press, 2007).
Friedrich, T. et al. High-throughput microarray technology in diagnostics of enterobacteria based on genome-wide probe selection and regression analysis. BMC Genomics 11, 591 (2010).
Zhang, T., Liu, G., Zhao, H., Braz, G. T. & Jiang, J. Chorus2: design of genome-scale oligonucleotide-based probes for fluorescence in situ hybridization. Plant Biotechnol. J. 19, 1967–1978 (2021).
Srinivasan, M., Sedmak, D. & Jewell, S. Effect of fixatives and tissue processing on the content and integrity of nucleic acids. Am. J. Pathol. 161, 1961–1971 (2002).
Thomsen, E. R. et al. Fixed single-cell transcriptomic characterization of human radial glial diversity. Nat. Methods 13, 87–93 (2016).
Alles, J. et al. Cell fixation and preservation for droplet-based single-cell transcriptomics. BMC Biol. 15, 44 (2017).
Ku, W.-C., Lau, W. K., Tseng, Y.-T., Tzeng, C.-M. & Chiu, S.-K. Dextran sulfate provides a quantitative and quick microarray hybridization reaction. Biochem. Biophys. Res. Commun. 315, 30–37 (2004).

ProBac-seq, a bacterial single-cell RNA sequencing methodology using droplet microfluidics and large oligonucleotide probe sets

Supplementary information

Supplementary Information

Supplementary Table 1

Supplementary Table 2

推荐阅读