High-throughput and high-accuracy single-cell RNA isoform analysis using PacBio circular consensus sequencing.
Zhuo-Xing ShiZhi-Chao ChenJia-Yong ZhongKun-Hua HuYing-Feng ZhengYing ChenShang-Qian XieXiao-Chen BoFeng LuoChong TangChuan-Le XiaoYi-Zhi LiuPublished in: Nature communications (2023)
Although long-read single-cell RNA isoform sequencing (scISO-Seq) can reveal alternative RNA splicing in individual cells, it suffers from a low read throughput. Here, we introduce HIT-scISOseq, a method that removes most artifact cDNAs and concatenates multiple cDNAs for PacBio circular consensus sequencing (CCS) to achieve high-throughput and high-accuracy single-cell RNA isoform sequencing. HIT-scISOseq can yield >10 million high-accuracy long-reads in a single PacBio Sequel II SMRT Cell 8M. We also report the development of scISA-Tools that demultiplex HIT-scISOseq concatenated reads into single-cell cDNA reads with >99.99% accuracy and specificity. We apply HIT-scISOseq to characterize the transcriptomes of 3375 corneal limbus cells and reveal cell-type-specific isoform expression in them. HIT-scISOseq is a high-throughput, high-accuracy, technically accessible method and it can accelerate the burgeoning field of long-read single-cell transcriptomics.
Keyphrases
- single cell
- high throughput
- rna seq
- induced apoptosis
- single molecule
- cell cycle arrest
- oxidative stress
- gene expression
- magnetic resonance
- dna methylation
- bone marrow
- signaling pathway
- magnetic resonance imaging
- genome wide
- poor prognosis
- stem cells
- optical coherence tomography
- cell death
- wound healing
- long non coding rna
- pi k akt