Sample Multiplexing Addendum

This page is a supplement to our post describing the distinctions between sample multiplexing and library multiplexing in single cell RNA-seq. Please see that page for more information.

Extensive use history in publications going back to 2018; well-supported by vendors like BioLegend
Greater flexibility in terms of how many samples can be multiplexed into a single GEM reaction
Requires compatible antibodies for your sample type
While it can greatly reduce reagent costs for the core RNA-seq library preparation, it does come with additional financial costs for antibodies and related reagents for staining the cells
Adds some new costs to the library preparation since it requires additional reagents to prepare a parallel library for the cell surface protein oligo tags (though overall you will likely still save a lot of money on reagent costs)
Requires additional time investment, both during protocol development (to optimize staining conditions) and during sample preparation for the single cell experiment (to stain and wash the cells)
BioLegend staining protocols assume an available input of 1,000,000+ cells, which may not be possible for certain sample types/experiments

While the sample preparation itself requires an additional fixation kit from 10X Genomics, the multiplexing is achieved without time/financial investment in developing a procedure to label the cells as in hashing experiments
The multiplexing barcodes are built into the assay and do not require the generation of secondary libraries as in hashing experiments
Only available for human and mouse samples (or possibly some very closely related species) without substantial time and monetary investment in development of custom probe sets
“Use it or lose it” multiplexing requires fully-multiplexed reactions (typically in groups of four or sixteen) in order to utilize full value of kit

On-Chip Multiplexing (OCM) in 3′ and 5′ Gene Expression Assays

From the submitting lab’s perspective, the OCM assays do not require any additional sample handling
The starting number of cells per sample required is much lower (though this fact can be deceptive – the required concentration in cells/uL is as high as in the standard/non-OCM assays)
Relatively cheap at ~$9,000 for a 16-reaction kit versus ~$24,000 for a 16-reaction kit of standard/non-OCM reagents (per April 2025 pricing)
Like with Flex, does not require the preparation of a secondary library containing multiplexing barcodes
Only supports a maximum of 5,000 cells recovered per sample. Samples can be loaded multiple times to increase this number, but this rapidly erodes the price benefit for the kit.
Like Flex, the multiplexing is “use it or lose it” and requires enough samples to make the run fully multiplexed in order to use the full value of the kit
The input volume per sample is capped at ~9 uL, so the OCM assays are not compatible with very low cell number “sort-and-load” projects