Single-cell RNA-seq analysis of mouse preimplantation embryos by third-generation sequencing.

• Main Authors: Xiaoying Fan, Dong Tang, Yuhan Liao, Pidong Li, Yu Zhang, Minxia Wang, Fan Liang, Xiao Wang, Yun Gao, Lu Wen, Depeng Wang, Yang Wang, Fuchou Tang
• Format: Article
• Language: English
• Published: Public Library of Science (PLoS) 2020-12-01
• Series: PLoS Biology
• Online Access: https://doi.org/10.1371/journal.pbio.3001017

The development of next generation sequencing (NGS) platform-based single-cell RNA sequencing (scRNA-seq) techniques has tremendously changed biological researches, while there are still many questions that cannot be addressed by them due to their short read lengths. We developed a novel scRNA-seq technology based on third-generation sequencing (TGS) platform (single-cell amplification and sequencing of full-length RNAs by Nanopore platform, SCAN-seq). SCAN-seq exhibited high sensitivity and accuracy comparable to NGS platform-based scRNA-seq methods. Moreover, we captured thousands of unannotated transcripts of diverse types, with high verification rate by reverse transcription PCR (RT-PCR)-coupled Sanger sequencing in mouse embryonic stem cells (mESCs). Then, we used SCAN-seq to analyze the mouse preimplantation embryos. We could clearly distinguish cells at different developmental stages, and a total of 27,250 unannotated transcripts from 9,338 genes were identified, with many of which showed developmental stage-specific expression patterns. Finally, we showed that SCAN-seq exhibited high accuracy on determining allele-specific gene expression patterns within an individual cell. SCAN-seq makes a major breakthrough for single-cell transcriptome analysis field.