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

Kang Y, McMillan I, Norris MH, Hoang TT.

Citation

Kang Y, McMillan I, Norris MH, Hoang TT. (2015) Single prokaryotic cell isolation and total transcript amplification protocol for transcriptomic analysis. Nature Protocols 10(7):974-984.

Abstract

Until 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 DNA with McrBC and DpnI restriction enzymes, single-stranded cDNA (ss-cDNA) ligation using T4 polynucleotide kinase and CircLigase, and polymerization of ss-cDNA to double-stranded cDNA (ds-cDNA) by �?�¦29 polymerase. This procedure takes �¢?�¼5 d, and sufficient amounts of ds-cDNA can be obtained from single-cell RNA template for further microarray analysis.