Single prokaryotic cell isolation and total transcript amplification protocol for transcriptomic analysis

U ntil recently, transcriptome analyses of single cells have been confined to eukaryotes. The information obtained from single-cell transcripts can provide detailed insight into spatiotemporal gene expression, and it could be even more valuable if expanded to prokaryotic cells. Transcriptome analysis of single prokaryotic cells is a recently developed and powerful tool. Here we describe a procedure that allows amplification of the total transcript of a single prokaryotic cell for in-depth analysis. This is performed by using a laser-capture microdissection instrument for single-cell isolation, followed by reverse transcription via Moloney murine leukemia virus, degradation of chromosomal DNANA with McrBC and DpnI restriction enzymes, single-stranded cDNANA (ss-cDNANA) ligation using T4 polynucleotide kinase and CircLigase, and polymerization of ss-cDNANA to double-stranded cDNANA (ds-cDNANA) by Phi 29 polymerase. This procedure takes similar to 5 d, and sufficient amounts of ds-cDNANA can be obtained from single-cell RNARNARNA template for further microarray analysis.