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

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Single Cell Total RNA Sequencing through Isothermal Amplification in Picoliter-Droplet Emulsion
Analytical Chemistry ( IF 6.7 ) Pub Date : 2016-11-01 00:00:00 , DOI: 10.1021/acs.analchem.6b02581
Yusi Fu ₁ , He Chen ₁ , Lu Liu _{1,

2} , Yanyi Huang _{1,

2,

3}

Affiliation

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.

中文翻译：

通过等温扩增在皮升-小滴乳液中的单细胞总RNA测序

普遍的单细胞RNA扩增和测序化学方法主要是通过使用oligo（dT）引物进行逆转录，来研究真核细胞中的聚腺苷酸化RNA。我们开发了一种新的RNA扩增方法“ easier-seq”，可从单个细胞中反转录和扩增带有和不带有聚腺苷酸尾巴的总RNA，以高效，可重复性和准确性进行转录组测序。通过将反转录的cDNA分子分布到1.5×10 ⁵在油中的水滴中，分别在这些65 pL反应器中的每一个中使用随机引物等温扩增cDNA。这种新方法通过减少实验步骤大大提高了单细胞RNA测序的简便性。同时，这种方法产生错误的机会较少，因此可以轻松维持单细胞测序的质量。另外，这种不依赖于聚腺苷酸尾巴的方法可以无缝地应用于原核细胞RNA测序。

更新日期：2016-11-01

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