Ensure High-Quality Single Cell Seq Runs with Moxi Instruments

Singe Cell Genomics

Relevant Instruments:

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According to 10x Genomics, a leader in single cell sequencing technology, the three most critical challenges to ensure a high-quality sequencing run are cell viability and integrity >70%, doublet detection and removal, and sample contamination.

All three of these can be addressed simultaneously on a single run with a Moxi V or Moxi GO II system from ORFLO Technologies. At a very affordable price, the Moxi V will give everything a user could need for their single cell sequencing experiments. If two-color viability stains, antibody-based immunoprofiling, or post-FACS GFP checking steps are important to the single cell workflow, the Moxi GO II can add these applications and is the instrument to choose.

Cell Counting + Viability

Fig. 1: Single cell library prep analysis for primary PBMCs on a Moxi GO II

The concentration of PBMCs was determined using the gating feature on the size data (x-axis) of the Moxi GO II’s intuitive post-run analysis software (Fig 1, left). Because lymphocytes and monocytes are significantly larger than debris and red blood cells (RBCs), it is simple to isolate them via size gating. The RBC concentration and overall sample contamination was determined using the same gating method with a separate gate for RBCs (3-6µm) (Fig. 1, right). Overall viability was assessed using propidium iodide (PI) and shows a healthy sample with a viability of 90.9%, significantly higher than the recommended 70% viability cutoff for 10x Genomics sequencing. With a relatively low RBC contamination percentage (18.7%) and the calculated concentration of PBMCs, it can be determined that this sample will yield good results if single cell sequencing is performed. However, the sample is a slightly higher concentration than the recommended maximum to ensure only a single cell is dispensed in each droplet (1,640 cells/µL vs recommended limit of 1,200 cells/µL). As such, this sample needs to be slightly diluted before starting a sequencing run.

Nuclei Counting

Fig. 2: Single cell library prep for nuclei isolated from a PBMC sample on a Moxi GO II.

This data was taken from a Moxi GO II user isolating nuclei from a PBMC sample for single cell sequencing. Accurate concentration calculations of nuclei when compared to the unlysed sample (Fig 2, right), in addition to easy identification of nuclei via PI staining, show the effectiveness of Moxi instruments in giving a fast, easy, reliable check of nuclei before sending it off for sequencing.

Aggregate Percentage Calculations

Fig. 3: Doublet calculations of a HEK293 single cell library prep on a Moxi GO II.

The data to the left was taken from a user doing a preparation of HEK293 cells for single cell analysis on a Moxi GO II. Viability measurements using propidium iodide (PI) shows high sample viability (Fig. 3). There is minimal sample contamination since very few events were counted at <3um. However, the doublet population is rather high for an optimal single cell sequencing run and this might cause downstream complications.

Post-FACS Purity Check

Fig. 4: Single cell library prep for ES-derived immature cardiomyocytes post-GFP sort on a Moxi GO II.

The above data was taken from a Moxi GO II user who performed FACS sorting on GFP-tagged immature cardiomyocytes before single cell sequencing. By using the Moxi GO II’s 525/40nm green channel (PMT1), the intact and properly sorted GFP-positive cardiomyocytes could easily be identified and counted (Fig. 4, left). Utilizing the second 561nm/LP orange/red channel at the same time, PI-based viability measurements can be performed in the same sample test (Fig. 4, left-middle). By using the Moxi post-run analysis software, the viability of the GFP-positive cells (GFP+, PI-) can be specifically determined (Fig. 4, right-middle). This allows for validation that the cells of interest have a high enough viability percentage to yield successful genomic data. Additionally, the aggregates and sample contamination can be calculated (Fig. 4, right), and with the combination of all of this data, the overall success of the potential sequencing run can be evaluated simply and effectively. In this case, while the sample is a slightly lower concentration than suggested (648 cells/µL vs recommended minimum of 700 cells/µL), is likely high enough quality to yield good sequencing results.

Summary

Comprehensive analysis of all the relevant parameters needed to ensure that a single cell sample is good to send off for sequencing can be done in just one test using a Moxi GO II or Moxi V. Both instruments come with unbeatable reproducibility of <5% CV in combination with the flexibility to adjust the post-run analysis software to accurately obtain data from a large number of cell types and fluorescent markers. Single cell library preparation has never been easier than with a Moxi instrument!