Sample Preparation for Single Cell Multiomics
The success of single cell experiments critically depends on effective sample preparation techniques. Here we discuss key strategies and practical tips for optimizing sample preservation, preparation, cleanup, cell counting, and quality control in single-cell multiomics experiments.
July 15, 2024
Sample Preservation
Cryopreservation
Cryopreservation of cells allows for long-term storage and enables batch processing of samples. Use cryoprotectants such as dimethyl sulfoxide (DMSO) to prevent ice crystal formation and cell damage during freezing. The suspension should be cooled down gradually in an isopropanol bath at -80C before moving it to liquid nitrogen for long term storage. Typically, nuclei don't survive the process of cryopreservation so only whole cells or tissues should be cryopreserved.
Flash Freezing
Whole tissues can be flash frozen in liquid nitrogen for long term stable storage. Wash the tissue, pat dry and chop into small (~2 mm size) pieces before freezing. Transfer the frozen tissue to vapor-phase liquid nitrogen for optimal long-term storage, avoid storing tissue at –80C for longer than 1–2 days. Flash freezing of tissues precludes whole cell analysis. Due to difficulties in isolating whole intact cells from frozen tissue, analysis of flash frozen samples should be limited to nuclei.
Fixation
Fixation of cells with chemical crosslinkers (e.g., 4% paraformaldehyde or in FFPE blocks ) preserves samples for long term storage. Optimize fixation conditions to minimize RNA degradation and preserve transcriptomic profiles. Fixed samples may not be suitable for poly A based transcriptomic readout due to the degraded RNA and may require the use of probe-based readouts.
Preservation at 4°C
Store samples at 4°C in appropriate buffer solutions to maintain cell viability and RNA integrity prior to processing. Avoid prolonged storage at 4°C to prevent RNA degradation, typical storage times recommended are between 24 and 72 hours.
Learn more how the BD® OMICS-Guard Sample Preservation Buffer protects both transcriptome and proteome information for up to 72 hours at 4C without the need of fixation or freezing.
Single Cell Isolation
Gentle dissociation for tissues
Use enzymatic or mechanical methods for gentle dissociation of tissues or cell aggregates into single-cell suspensions. Optimize digestion conditions to achieve maximal cell yield while minimizing cell stress and RNA degradation. Commercially available tissue dissociation devices or individual lab-designed dissociation techniques can be used.
Check out the Worthington Tissue Dissociation Guide for protocols for your tissue type.
Sorting
Employ techniques such as fluorescence-activated cell sorting (FACS) for cleaning up the single cell suspension by removing debris and multiplets or to enrich for cells of interest. Ensure proper calibration and optimization of sorting parameters to minimize cell stress. It is a good idea to count and check viability of single cells after sorting.
Explore the range of cell sorters from BD Biosciences to rapidly sort your cells for subsequent single cell multiomics analysis.
Bead-based cleanup
Bead-based methods use beads that recognize specific moieties on the cell surface. These beads sequester targeted cells, resulting in a flow-through that is devoid of that particular population.
The beads are small enough that they do not interfere with most single cell multiomics methods if positively selected. Cells should be thoroughly washed and resuspended in an appropriate buffer before use.
Density gradient centrifugation
Density gradient centrifugation is commonly employed to fractionate PBMCs or isolate plasma from blood cells. Additionally, it effectively removes large debris and cell aggregates from single cell or single nuclei suspensions. Some of the commonly used density gradient centrifugation techniques include Iodixanol, sucrose, Percoll and Ficoll.
Quality control
Washing
Simple washes are great for removing debris, dead cells, interfering reagents and ambient RNA. To maximize cell viability, optimize centrifugation speeds to prevent harm to the cells (smaller moieties need higher speeds) and maintain at 40C in the centrifuge. Pipette gently with a wide bore pipette.
Filtering
Filtering cell suspensions using an appropriate cell strainer is beneficial for eliminating large cell clumps and aggregated debris, thereby ensuring optimal performance. Select a strainer with a pore size larger than the maximum cell diameter in the sample, but still small enough to capture larger clumps.
Buffer check
Choose appropriate wash & resuspension buffers (sample type and assay dependent) so as not to affect downstream reactions. DNAse, EDTA, high levels of serum (BSA,FBS), surfactants (Tween-20) should be avoided in general. Nuclei suspensions should always be maintained in a buffer containing RNAse inhibitor if intended to be used for RNA-Seq.
Cell Counting and Viability
Use a small cell aliquot on automated cell counters or hemocytometers for accurate cell counting and viability assessment. Include dead cell exclusion dyes (e.g., propidium iodide, ethidium bromide) to discriminate live from dead cells and calculate cell concentration accurately. Dead and dying cells compromise your single cell data by creating high background, so aim for at least 70% viability.
Explore how the BD® Rhapsody Scanner provides a visual QC system that can be combined with a microwell-based single-cell partitioning system without the need to buy third party cell counters.
BD Biosciences provides high-quality single-cell multiomics reagents for use in research settings.
