Please use this identifier to cite or link to this item: http://hdl.handle.net/10125/40214

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

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Title: Single prokaryotic cell isolation and total transcript amplification protocol for transcriptomic analysis
Authors: Kang, Yun
McMillan, Ian
Norris, Michael H.
Hoang, Tung T.
Issue Date: Jul 2015
Related To: http://www.nature.com/nprot/journal/v10/n7/full/nprot.2015.058.html
http://www.ncbi.nlm.nih.gov/pubmed/?term=%22Single+prokaryotic+cell+isolation+and+total+transcript+amplification+protocol+for+transcriptomic+analysis%22
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 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 utilizing 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, ss-cDNA ligation using T4 polynucleotide kinase and CircLigase, and polymerization of ss-cDNA to ds-cDNA by ϕ 29 polymerase. This procedure takes ~5 days, and sufficient amounts of ds-cDNA can be obtained from single cell RNA template for further microarray analysis.
Pages/Duration: 22
URI/DOI: http://hdl.handle.net/10125/40214
DOI: 10.1038/nprot.2015.058
Appears in Collections:Department of Microbiology Faculty & Researcher Works



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