Single Cell Genomics
Higher RNA Yield, Better Gene Expression, More Consistent Data
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Techniques for Single Cell RNA-Sequencing (scRNA-seq)
Single cell genomics methodologies, such as single-cell RNA sequencing (RNA-seq), have revolutionized the study of cellular heterogeneity and helped to identify and characterize different cell types and cell subtypes. There are two primary approaches to the single-cell sequencing workflow: droplet-based and plate-based.
Droplet-Based RNA-Sequencing
Droplet-based RNA-seq (such as 10x Genomics’ Chromium system) can be used on live cells or nuclei from tissue samples. It is a cost-effective and high throughput single-cell RNA-sequencing (scRNA-seq) approach that can profile up to tens of thousands of cells per assay. The droplet-based RNA-sequencing approach requires high quality cell or nuclei suspensions, with minimal aggregates and debris. Isolation of individual cells or high quality nuclei, removal of dead cells, debris and clumps from the sample and improved overall quality of single-cell sequencing can be readily achieved using Bio-Techne’s Cell Sorters and Single Cell Dispensers in the bulk sorting mode.
Plate-Based RNA-Sequencing
Plate-based RNA-seq differs from droplet-based RNA-seq in that it entails the sequencing of single-cell transcriptomes whereas droplet-based single cell sequencing may be limited to covering only the 3′ portion of transcripts. Plate-based RNA-seq therefore provides more complete profiling of the transcriptome and superior detection of gene expression. Traditional fluorescence-activated cell sorting (FACS) is too harsh for isolating single cells in plates, as it can harm cell integrity and inducinduce stress-related transcriptional changes. Bio-Techne’s cell sorters operate at low pressure, dramatically improving library quality for plate-based transcriptome sequencing in bioinformatics, offering a useful alternative to FACS sorting.
Transform Your Single Cell Genomics Workflow
The illustration above (workflow A) outlines a droplet-based workflow, in which a fresh frozen tissue sample is used to prepare a nuclei suspension, stained for nucleic acid, and loaded on the Bio-Techne Cell Sorter and Single Cell Dispenser to bulk sort fluorescently labeled intact nuclei; debris and degraded nucleic acid are left behind. Droplet-based cDNA synthesis and sequencing can then be performed using the purified nuclei.
The illustration above (workflow B) outlines a plate-based workflow. A cell suspension is prepared, which is loaded into a Bio-Techne microfluidic cartridge, and cells are then dispensed into 96- or 384-well plates pre-loaded with lysis buffer, using Bio-Techne's single cell dispenser instrument as an alterative to fluorescence activated cell sorting (FACS). Following this single cell isolation step, individual cells are ready for multiple downstream genomic applications, such as single nuclei RNA sequencing (snRNA-seq) or single-cell RNA sequencing (scRNA-seq).
Gentle
Gentle cell sorting (< 2 psi) preserves cell integrity for single cell sequencing (RNA-seq)
Fast
Dispenses individual cells into 96-well plate in 1 min, or 384-well plate in 6 min
Easy
Easy to use; no need for in-house technical experts; easily isolate rare cell types (<0.1% of population)
Flexible
Works with cell density ranging from 100 cells/mL to 150M cells/mL
Affordable
Reduced reaction volume, cuts reagent costs by 90%
Efficient
Bio-Techne’s Cell Sorters and Single Cell Dispensers are highly efficient and consistent at sorting and dispensing single cells (80-90% singlets with mammalian cells - see image right).
Bio-Techne Cell Sorters versus FACS
The figure shows a comparison of Bio-Techne's Hana cell sorter vs a fluorescence-activated cell sorting instrument for sorting cells to use in single cell sequencing protocols. Bio-Techne’s Hana Cell Sorter and Single Cell Dispenser was used to sort cells for single-cell RNA sequencing (scRNA-seq) alongside a traditional FACs sorting instrument. Cell populations 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.
The figure shows a comparison of stress-related gene expression with the Hana cell sorter versus a FACs sorting system. Cells sorted with a FACS sorting 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.
Improving Your 10X Genomic Workflows Using the Pala Cell Sorter from Bio-Techne
Single cell sequencing methods have emerged as powerful tools for identification of heterogeneous cell types. In this application note, we compared single-nucleus RNA sequencing (snRNA-seq) data from dissociated nuclei using traditional methods compared to sorting nuclei using Bio-Techne’s Pala benchtop cell sorter. The sequencing data showed the Pala system was able to sort nuclei to successfully clear debris from brain and kidney dissociated tissue, recover more cells for downstream 10X genomic workflows, and displays cleaner sequencing data with higher gene recognition and characterization of diverse cell types. The results serve as a reference for researchers seeking to improve their genomic workflow and uncover results that may have been hidden using traditional methods.
Citations
Coexpression network and trans-activation analyses of maize reproductive phasiRNA loci. Zhan, J., et al. (2023), Plant J, 113: 160-173. https://doi.org/10.1111/tpj.16045
FX-Cell: Quantitative cell release from fixed plant tissues for single-cell genomics. Marchant, D.B., et al. bioRxiv 2021.10.11.463960; doi:https://doi.org/10.1101/2021.10.11.463960
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