SorTn-seq: a high-throughput functional genomics approach to discovering regulators of bacterial gene expression

This protocol describes a high-throughput sequencing approach to identifying regulators of bacterial gene expression based on transposon insertion mutagenesis in a strain harboring a fluorescent reporter. We recently developed a high-throughput functional genomics approach, named 'SorTn-seq', to identify factors affecting expression of any gene of interest in bacteria. Our approach facilitates high-throughput screening of complex mutant pools, a task previously hindered by a lack of suitable techniques. SorTn-seq combines high-density, Tn5-like transposon mutagenesis with fluorescence-activated cell sorting of a strain harboring a promoter-fluorescent reporter fusion, to isolate mutants with altered gene expression. The transposon mutant pool is sorted into different bins on the basis of fluorescence, and mutants are deep-sequenced to identify transposon insertions. DNA is prepared for sequencing by using commercial kits augmented with custom primers, enhancing ease of use and reproducibility. Putative regulators are identified by comparing the number of insertions per genomic feature in the different sort bins, by using existing bioinformatic pipelines and software packages. SorTn-seq can be completed in 1-2 weeks and requires general microbiology skills and basic flow cytometry experience.

Automated summary

This protocol describes a high-throughput sequencing approach called 'SorTn-seq' for identifying regulators of bacterial gene expression through transposon mutagenesis and fluorescence-activated cell sorting. The method enables efficient screening of mutants with altered gene expression and can be completed in approximately 1-2 weeks with basic microbiology skills and flow cytometry experience.