Single-cell RNA sequencing

Resolve isoform diversity at the single-cell level

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. 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

Explore recommendations for all SMRT sequencing applications

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.

Sequencing

Use HiFi reads on the Sequel II or IIe systems to generate 3 million full-length single-cell molecules from 1 SMRT Cell 8M to obtain 1,000 UMIs for 3,000 single cells**

**Read lengths, number of reads, data per SMRT Cell 8M and other sequencing performance results vary based on sample quality/type and insert size, among other factors

Data analysis

Identify, classify, and filter full-length isoforms at the single-cell level

Analyze HiFi reads which allow accurate single-cell barcode and UMI identification
Use the single-cell Iso-Seq analysis tools on GitHub to output high-quality, full-length transcript FASTA sequences per UMI, with no assembly required, to characterize transcripts variants for each cell

TECHNOLOGY DEVELOPMENT SPOTLIGHT

Mas-ISO-Seq method delivers > 15X throughput through concatenation

Our technology is constantly evolving. MAS-ISO Seq data takes advantage of long and accurate HiFi read lengths to increase Iso-Seq throughput by > 15-fold through multiplexed arrays of cDNA molecules.

Watch the MAS-ISO-Seq talk at ASHG or read the 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 et al. (2021) A spatially resolved brain region- and cell type-specific isoform atlas of the postnatal mouse brain, Nature Communications