Improving cell recovery in single cell RNA sequencing

High-quality single-cell RNA sequencing (scRNA-seq) starts with efficient, gentle, tissue dissociation—but inconsistent yields, low cell viability, and lengthy prep times can slow you down. The right tissue disaggregation method ensures high cell recovery, minimal stress on individual cells, and reproducible results.

Our latest application note explores how advanced tissue disaggregate streamlines single-cell sample preparation, delivering high-viability cell suspensions optimized for scRNA-sequencing and other single-cell analysis applications.

In our study we use single-cell RNA seq quality metrics to understand how the VIA Extractor™ tissue disaggregator performs compared to the Miltenyi Biotec gentleMACS dissociator in fresh mouse liver tissue dissociation.

Our findings:

• Significantly more cells recovered with the VIA Extractor tissue disaggregator.
• A significantly higher number of mitochondrial reads with the gentleMACS dissociator, potentially indicating apoptic, stressed, or low-quality cells.
• Direct comparison through overlay of the cell clusters, suggesting that dissociation using the VIA Extractor tissue disaggregator paired with the HIVE™ scRNAseq is gentle enough to allow the recovery of a more cells.

Clinical and academic researchers are driven by a need to understand the true biology of living organisms. To achieve this, organs, biopsies, tumors, and other cell types and tissue materials are collected from animal models and human patients and transported to the research labs.

To fully comprehend the role of every cell, single cell sequencing methods have rapidly gained momentum over the last decade. However, delays caused by tissue transportation and the harsh and lengthy sample preparation processes can subject the tissue sample to additional stress. These conditions can alter the transcriptional profiles during the cell analysis, resulting in data that do not accurately represent the sample in situ. To address these issues, we pair our VIA Extractor tissue disaggregator workflow with the HIVE scRNAseq solution from Honeycomb Biotechnologies (HCB). The HIVE workflow enables the capture, storage, and sequencing of single cells, providing a rapid yet gentle sample preparation protocol followed by the preservation of single cells, which can be processed for single cell sequencing at a convenient time.

Sample preparation and scRNA-Seq analysis: A step-by-step approach

Whole livers were collected from three male Crl:CD1 (ICR) mice and transported to laboratories on cool packs for processing. The right lobe of the liver was used for the studies and divided into two portions. One portion was dissociated using the VIA Extractor tissue disaggregator, and the other with the gentleMACS dissociator following the manufacturer’s instructions. Both samples used the Miltenyi Biotec Liver Dissociation Kit enzymes to minimize variability. All six samples (3 × gentleMACS dissociator and 3 × VIA Extractor tissue disaggregator) were processed equally downstream of tissue dissociation to single-cell sequencing. Following automated tissue dissociation, the samples were filtered using a 100 μm cell strainer. Red blood cell lysis was performed using the Miltenyi Biotec Red Blood Cell lysis following the manufacturer’s recommendations. Cells were counted using the NucleoCounter NC-200 automated cell counter, and 10 000 cells were loaded into a HIVE collector for each sample. The cell-loaded HIVEs were incubated for 30 minutes, allowing individual cells to settle gently into the picowells pre-loaded with 3 barcoded mRNA-capture beads. A wash was undertaken to remove residual media before adding the proprietary cell preservation solution (HCB) and storing the samples at -20°C for five days. The cell-loaded HIVEs were shipped to HCB on dry ice, processed, cDNA libraries prepared, and then single-cell sequencing analysis took place. Count matrices were generated by HCB using BeeNet software. The scRNA matrix data were analyzed by HCB using a seurat-based workflow (1).

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By implementing these strategies, you can improve cell recovery, reduce prep time, and enhance the quality of your sequencing data. Take the next step and download the full application note to unlock the details.