SorTn-seq: a high-throughput functional genomics approach to discovering regulators of bacterial gene expression.
Leah M SmithSimon A JacksonPaul P GardnerPeter C FineranPublished in: Nature protocols (2021)
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.
Keyphrases
- single cell
- high throughput
- gene expression
- rna seq
- high density
- flow cytometry
- dna methylation
- wild type
- single molecule
- transcription factor
- genome wide
- copy number
- crispr cas
- poor prognosis
- living cells
- machine learning
- quantum dots
- energy transfer
- cell free
- label free
- mesenchymal stem cells
- cell therapy
- data analysis
- bone marrow
- binding protein
- nucleic acid