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. Researchers from Peking University have developed 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-picoliter reactors separately. This new method greatly improves the ease of single-cell RNA sequencing by reducing the experimental steps. 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.

rna-seq

Fu Y, Chen H, Liu L, Huang Y. (2016) Single cell total RNA sequencing through isothermal amplification in picoliter-droplet emulsion. Anal Chem [Epub ahead of print]. [abstract]

One comment

  1. This protocol has a non negligible drawback that a hugh amount of rRNA reads are presents in the raw data, so it’s super-costly and also somehow masks some true biological info since the lack of enough sequencing depth.

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