Single Cell Total RNA Sequencing through Isothermal Amplification in Picoliter-Droplet Emulsion

Prevalent single cell RNA amplification and sequencing chemistries mainly focus on polyadenylated RNAs in eukaryotic cells by using oligo­(dT) primers for reverse transcription. We develop a new RNA amplification method, “easier-seq”, to reverse transcribe and amplify the total RNAs, both with and w...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2016-11, Vol.88 (22), p.10795-10799
Main Authors: Fu, Yusi, Chen, He, Liu, Lu, Huang, Yanyi
Format: Article
Language:English
Subjects:
Amplification
Biochemistry
Cells
Deoxyribonucleic acid
Distributing
DNA
Droplets
Efficiency
Gene sequencing
Molecules
Reactors
Reproducibility
Ribonucleic acid
Ribonucleic acids
RNA
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Summary:Prevalent single cell RNA amplification and sequencing chemistries mainly focus on polyadenylated RNAs in eukaryotic cells by using oligo­(dT) primers for reverse transcription. We develop a new RNA amplification method, “easier-seq”, to reverse transcribe and amplify the total RNAs, both with and without polyadenylate tails, from a single cell for transcriptome sequencing with high efficiency, reproducibility, and accuracy. By distributing the reverse transcribed cDNA molecules into 1.5 × 105 aqueous droplets in oil, the cDNAs are isothermally amplified using random primers in each of these 65-pL reactors separately. This new method greatly improves the ease of single-cell RNA sequencing by reducing the experimental steps. Meanwhile, with less chance to induce errors, this method can easily maintain the quality of single-cell sequencing. In addition, this polyadenylate-tail-independent method can be seamlessly applied to prokaryotic cell RNA sequencing.
ISSN:0003-2700
1520-6882
DOI:10.1021/acs.analchem.6b02581