Workflow: single-cell transcriptomics
Obtaining full-length isoforms from single cells with nanopore sequencing
Characterisation of transcriptomic differences between individual single cells, or spatially across tissues, has provided insight into how the genome is utilised for specialised functions, such as neurological disorders and cancer. However, the majority of these studies recover limited isoform and single nucleotide polymorphism (SNP) content due to the limited read length of short-read sequencing. With the latest nanopore sequencing technology, it is now
simple to get isoform-level expression from full-length cDNAs at the throughput needed to analyse thousands of cells.
RNA from single cells can be prepared using the 10x Genomics microfluidics-based Chromium platform, which produce barcoded, full-length cDNA from individual cells. 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 and isoform switching, such as during development. In addition, long nanopore reads enable the detection of both 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, with library prep in approximately three hours.