High-throughput single-cell transcriptomics of bacteria using combinatorial barcoding

doi:10.1038/s41596-024-01007-w

High-throughput single-cell transcriptomics of bacteria using combinatorial barcoding

2024-06-17

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DOI：10.1038/s41596-024-01007-w

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Microbial split-pool ligation transcriptomics (microSPLiT) is a high-throughput single-cell RNA sequencing method for bacteria. With four combinatorial barcoding rounds, microSPLiT can profile transcriptional states in hundreds of thousands of Gram-negative and Gram-positive bacteria in a single experiment without specialized equipment. As bacterial samples are fixed and permeabilized before barcoding, they can be collected and stored ahead of time. During the first barcoding round, the fixed and permeabilized bacteria are distributed into a 96-well plate, where their transcripts are reverse transcribed into cDNA and labeled with the first well-specific barcode inside the cells. The cells are mixed and redistributed two more times into new 96-well plates, where the second and third barcodes are appended to the cDNA via in-cell ligation reactions. Finally, the cells are mixed and divided into aliquot sub-libraries, which can be stored until future use or prepared for sequencing with the addition of a fourth barcode. It takes 4 days to generate sequencing-ready libraries, including 1 day for collection and overnight fixation of samples. The standard plate setup enables single-cell transcriptional profiling of up to 1 million bacterial cells and up to 96 samples in a single barcoding experiment, with the possibility of expansion by adding barcoding rounds. The protocol requires experience in basic molecular biology techniques, handling of bacterial samples and preparation of DNA libraries for next-generation sequencing. It can be performed by experienced undergraduate or graduate students. Data analysis requires access to computing resources, familiarity with Unix command line and basic experience with Python or R. Through four rounds of combinatorial barcoding, this low-cost and high-throughput single-cell RNA sequencing method enables transcriptomic analysis of individual bacterial cells and the detection of phenotypically distinct subpopulations.Conditions for cell wall digestion, membrane permeabilization and the barcoding procedure are optimized to profile tens of thousands of bacterial cells in a single bench-based barcoding experiment without the need for a dedicated instrument. Single-cell transcriptomics of bacteria is challenging. microSPLiT is a high-throughput method for single-cell RNA sequencing of both Gram-positive and Gram-negative bacteria using combinatorial barcoding without the need for specialized equipment.

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GENE-EXPRESSION

RNA

High-throughput single-cell transcriptomics of bacteria using combinatorial barcoding

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