Single cell genomics has revolutionized the study of cellular heterogeneity. New approaches, including single cell RNA sequencing (RNA-seq), help identify and define cell subtypes. Single cell RNA-seq workflows fall into two primary methodological categories: droplet-based and plate-based.
Droplet-based RNA-seq (such as 10x Genomics’ Chromium system) is a high throughput and cost-effective single cell RNA-seq approach that can profile up to tens of thousands of cells per assay. It can be used on live cells or nuclei from tissue samples. It is critical to obtain high quality cell or nuclei suspension with minimal aggregates and debris for droplet-based RNA-seq. Using the bulk sorting mode, Namocell’s Single Cell Dispensers can be used to isolate live cells or high quality nuclei, removing dead cells, debris and clumps from the sample and improving overall RNA-seq data quality.
Unlike droplet-based RNA-seq that can be limited to covering only the 3′ portion of transcripts, plate-based RNA-seq entails sequencing full-length transcripts from single cells partitioned in microplates. Thus, plate-based RNA-seq approach offers the advantages of more complete profiling of the transcriptome and better detection of rare or lowly expressed genes. To isolate single cells in plates, traditional FACS instruments are too harsh, damaging cell integrity and inducing stress-related transcriptional changes. Namocell’s Single Cell Dispensers operate at low pressure, dramatically improving library quality for plate-based single cell RNA-seq.
In a droplet-based workflow (above) using fresh frozen tissue sample, a nuclei suspension is prepared, stained for nucleic acid, and loaded on the Namocell Single Cell Dispenser to bulk sort fluorescently labeled intact nuclei, leaving debris, and degraded nucleic acid behind. The purified nuclei can proceed with droplet-based cDNA synthesis and sequencing.
In plate-based workflow (below), a cell suspension is prepared, loaded into a Namocell’s microfluidic cartridge, and dispensed into 96- or 384-well plates pre-loaded with lysis buffer. Following single cell isolation, cells are ready for multiple downstream genomic applications, including single cell RNA-seq.
Gentle sorting (< 2 psi) preserves cell integrity
Dispense single cells into 96-well plate in 1 min, or 384-well plate in 6 min
Easy to use, no need for in-house technical experts
Easily isolate rare cells (<0.1% of population)
Work with cell density ranging from 100 cells/mL to 150M cells/mL
Reduce reaction volume, cut reagent costs by 90%
Namocell’s HanaTM Single Cell Dispenser was used to sort cells for single cell RNA-seq alongside a traditional FACS instrument. Cells sorted with Hana showed markedly higher numbers of reads and genes detected per cell (post-filtering), higher mapping rates, and lower fractions of mitochondrial reads.
Cells sorted with a FACS instrument showed induction of cell stress genes such as Ubiquitin C (Ubc), likely owing to high-pressure sorting conditions (20-70 psi). Cells sorted on the low-pressure Hana system (<2 psi) showed much lower expression of these stress-related transcripts.