Workflow: single-cell transcriptomicsLiterature
Date: 17th October 2022
Obtaining full-length isoforms from single cells with long nanopore sequencing reads
Sequencing cDNA at single-cell resolution can reveal transcriptomic differences between individual cells. The ability to characterise single-cell gene expression has provided an insight into how the different tissues and cell types in an organism utilise the genome for specialised functions, and has elucidated mechanisms of disease.
RNA from single cells can be prepared using the 10x Genomics microfluidics-based Chromium platforms, which produce barcoded, full-length cDNA from individual cells. However, traditional short-read sequencing of these single-cell libraries typically yields only around 90 bp of sequence aligned to one end of each transcript. This limited representation of each transcript makes it difficult to quantify isoform-level expression.
Nanopore sequencing is compatible with the 10x Genomics sample preparation approach and can be used to sequence full-length transcripts and splice variants, providing detail on isoform diversity or isoform switching, such as during development. In addition, long nanopore reads enable the detection of both single nucleotide polymorphisms (SNPs) for RNA-based genotyping and gene fusions that are often associated with cancer.
Here, we present a complete workflow for single-cell transcriptome analysis from 10x Genomics cDNA libraries, using the PromethION device.