Confidently Resolve Cellular Heterogeneity
Differences in isoform expression and relative abundance among individual cells can be an important clue in understanding how populations of cells interact to function as a complex system. Whether you are exploring the immune system, the brain, or tumor environment, only highly accurate long reads (HiFi reads) can provide unbiased, direct detection of isoforms in your single-cell study.
Resolve isoform diversity at the single-cell level
HiFi reads deliver the high accuracy and long reads needed to capture intact isoform information without assembly or complicated algorithms, including at the single cell level. Single-cell RNA sequencing using the Iso-Seq method allows you to:
- Generate full-length transcript isoforms that can be confidently assigned to individual cells
- Move beyond 3’ gene counting to include TSS, polyA, and complete exon connectivity data to deepen your understanding of cell type differences
- Distinguish cell types that play unique roles in complex systems like the immune system
- Understand how alternative splicing of critical genes drives function in tissues like the brain
Workflow: From RNA to full-length transcripts at a single-cell level
|Sample & Library Preparation|
|Prepare full-length transcripts from single-cell RNA preparations using your preferred platform|
- Enrich for single-cell cDNA using a single-cell sorting platform that generates full-length cDNA*
- Template switch oligo (TSO)-based cDNA synthesis methods are recommended
- The final single-cell cDNA product consists of 5’ primer, transcript, poly-A tail, unique molecular index (UMI), cell barcode and 3’ primer
- To generate matching short-read data, save 5% of the material
- Additional PCR cycles can be added if necessary
- Start library preparation with at least 160 ng of input cDNA (post-single-cell platform PCR reaction) for 1-2 SMRT Cell 8M
- More starting material will be required for sequencing multiple SMRT Cells 8M
- Prepare libraries with the SMRTbell Express Template Prep Kit 2.0 in one day
* Number of usable reads, containing the UMI and cell barcode, vary by single-cell platform. Any platform that generates full-length cDNA is compatible with the single-cell Iso-Seq sequencing workflow.
|Simplify sample prep workflow and reduce project costs by using the Sequel Systems|
- Use HiFi reads on the Sequel II or IIe Systems to generate 3 million full-length reads from one SMRT Cell 8M to obtain ~1,000 unique molecules for 3,000 single cells**
- Use 24 hr movies with 2 hrs pre-extension time
- For human samples, run up to 240 SMRT Cell 8M/year
**Read lengths, number of reads, data per SMRT Cell, and other sequencing performance results vary based on sample quality/type and insert size, among other factors.
|Identify, classify, and filter full-length isoforms at the single-cell level|
Application Brief: Learn more about these best practices for single-cell RNA sequencing
Spotlight: HIT-scISOseq enables high throughput, high accuracy, single-cell sequencing of full-length transcripts
HIT-scISOseq yields >10 million high-accuracy full-length isoforms in a single SMRT Cell 8M on the Sequel II System for high-throughput single-cell isoform sequencing. By combining full-length cDNA capture with biotinylated PCR primers and a novel head-to-tail concatenation step, HIT-scISOseq provides eight times more data output than the standard single-cell RNA sequencing protocol.
Zheng, Y.-F., et al. (2020) HIT-scISOseq: High-throughput and high-accuracy single-cell full-length isoform sequencing for corneal epithelium. bioRxiv Preprint.
Spotlight: Gene-wise comparisons reveal more differences between mouse neuronal cell types than exon pairwise comparisons
Using full-length isoform information, including transcription start site (TSS) and polyA data, 395 genes show significantly different expression in the hippocampus versus prefrontal cortex cells, whereas only 31 genes met the same criteria using short-read data. In the above figure, a 6nt microexon in Nsfl1c (orange) is preferentially expressed in the hippocampus across neuronal and glial cell types but is absent in the same pre-frontal cortex cell types.
Joglekar, A., et al. (2020) Cell-type, single-cell, and spatial signatures of brain-region specific splicing in postnatal development. bioRxiv Preprint.
To learn more about how to use single-cell RNA sequencing in your research, contact us.
- Poster: Tseng, E. et al. (2019) Single cell isoform sequencing (scIso-Seq) identifies novel full-length mRNAs and cell type-specific expression
- Wenger, Aaron and Tseng, Elizabeth (2020) ASHG CoLab: PacBio HiFi reads for comprehensive characterization of genomes and single-cell isoform expression
- Korlach, Jonas (2020) ASHG PacBio Workshop: Latest product and application updates
- Tilgner, Hagen (2020) ASHG PacBio Workshop: Single-cell isoform analysis of the nervous system
- Korlach, Jonas and Graves-Lindsay, Tina and Vierra, Michelle (2020) PacBio Workshop: Understanding the biology of genomes with HiFi sequencing
- Underwood, Jason (2019) AGBT Presentation: Single cell isoform sequencing (scIso-Seq) identifies novel full-length mRNAs and cell type-specific expression
- SMRT Sequencing Brochure: Delivering highly accurate long reads to drive discovery in life science (2020)
- Application Brief: Single-cell RNA sequencing with HiFi reads – Best Practices (2020)
- Application Brochure: What can you do with one SMRT Cell? (2020)
- Product Brochure: Sequel IIe System – Sequencing evolved (2020)
- Informational Guide: Understanding accuracy in DNA sequencing (2020)
- Application Brochure: HiFi reads for highly accurate long-read sequencing (2020)
- Informational Guide: What’s the value of sequencing full-length RNA transcripts? (2020)