학술논문
Single prokaryotic cell isolation and total transcript amplification protocol for transcriptomic analysis
Document Type
Report
Source
Nature Protocols. July 1, 2015, p974, 11 p.
Subject
Language
English
ISSN
1754-2189
Abstract
INTRODUCTION The ability to isolate a single prokaryotic cell and to analyze its transcriptome is a powerful new tool that can help to elucidate unique behaviors in bacteria that are [...]
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 ~5 d, and sufficient amounts of ds-cDNA can be obtained from single-cell RNA template for further microarray analysis.
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 ~5 d, and sufficient amounts of ds-cDNA can be obtained from single-cell RNA template for further microarray analysis.