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

Abstract

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.

BCAS2 is involved in alternative splicing and mouse oocyte development

“…The result showed that γH2AX foci were present in the nuclei in both Bcas2‐cKO Vasa and control oocytes, and the number of γH2AX foci was comparable between these two groups (Figure S3A,B), suggesting that DNA damage levels might be almost normal in Bcas2‐cKO Vasa oocytes. To unravel whether BCAS2 is involved in mRNA splicing in oocytes, we performed the latest developed method of SCAN‐seq (single‐cell amplification and sequencing of full‐length RNAs on the Nanopore platform), a new tool for single‐cell RNA sequencing using third‐generation sequencing technology 10 …”

“…To unravel whether BCAS2 is involved in mRNA splicing in oocytes, we performed the latest developed method of SCAN-seq (single-cell amplification and sequencing of full-length RNAs on the Nanopore platform), a new tool for single-cell RNA sequencing using third-generation sequencing technology. 10 Four oocytes from the control and Bcas2-cKO Vasa PD10 ovaries were collected and subjected to the library construction of SCAN-seq from each group. SCAN-seq detected 11 894 genes in all of these samples (RPG10K > 0.01).…”

“…Eventually, these oocytes are arrested at the metaphase of meiosis II (MII) after ovulation 6,8,9 . During this process, ~50% of genome‐wide genes are transcribed and their mRNA accumulates in mouse oocytes 10 . However, how the expression of these genes is regulated in mouse oocytes remains largely unclear.…”

Methods and platforms for analysis of nucleic acids from single-cell based on microfluidics

“…For single-cell RNA analysis, the problem with short-read length still needs to be further improved. To address this issue, Fan et al ( 2020 ) developed a single-cell RNA sequencing technology based on the third-generation sequencing, e.g., nanopore sequencing. With this platform, 27,250 unannotated transcripts from 9338 genes were analyzed and identified.…”

Possible Role of Accessory Proteins in the Viral Replication for the 20I/501Y.V1 (B.1.1.7) SARS CoV-2 Variant

“…This system can be further modified to understand gene expression under specific conditions or at particular time [9,10]. Targeted sequencing was used to identify relevant pharmacogenes to facilitate precise medicine [11]and single-cell analysis was used to analyze allele specific gene expression different developmental stages in mouse embryo [12]. Further transcriptome network analysis has also been used to understand the severity of asthmatic condition in children [13].…”