Question: Can I pool less than 4 or 16 samples when using the Chromium Fixed RNA Profiling Reagent Kits for Multiplexed Samples?
Answer: We recommend using either 4 or 16 samples for multiplexing with the Fixed RNA Profiling Assay (i.e., Single Cell Gene Expression Flex assay) to maximize the multiplexing efficiency of the kits. However, other configurations are possible.
This illustration provides an overview of multiplexing configurations using the Chromium Fixed RNA Kit, Human Transcriptome, 4 rxns x 4 BC PN-1000475. The same principle can be applied when using the Chromium Fixed RNA Kit, Human Transcriptome, 4 rxns x 16 BC PN-1000476; Max # of cells per Probe Barcode is 8,000 cells.
Subpooling
If fewer samples than the number barcodes (BC) available are used (e.g., less than 4 or 16), then it is possible to subpool a sample across multiple barcodes to use all barcodes in a single kit. Sub-pooling one sample over multiple Probe Barcodes will allow the capture of more cells from that sample, with a lower undetected multiplet rate.
This illustration provides an overview of multiplexing configurations using the Chromium Fixed RNA Kit, Human Transcriptome, 4 rxns x 4 BC PN-1000475. The same principle can be applied when using the Chromium Fixed RNA Kit, Human Transcriptome, 4 rxns x 16 BC PN-1000476; Max # of cells per Probe Barcode is 8,000 cells.
Multiplexing < 4 or < 16 samples
It is possible to multiplex fewer than 4 or 16 samples, for example if a sample is lost during hybridization or post-hybridization washing, or if fewer than the maximum number of samples is desired. In such cases, we recommend multiplexing or pooling the remaining samples post-hybridization and continuing with the protocol. Consult Fixed RNA Profiling for Multiplexed Samples - Pooling Workbook (Document CG000565) for guidance on such alternate multiplexing strategies. This worksheet can also be used for calculating sample volumes for multiplexing/pooling.
If multiplexing less than 4 or 16 samples, there may be extra unused barcodes if deciding not to subpool samples. 10x does not recommend or support mixing reagents and barcodes from different kit lots. If combining barcodes from different kits for alternative configuration, it will be important to keep track of which probe barcodes are used in a plex, so that there is no overlap. Additional kit(s) will need to be purchased to utilize the unused probe barcodes in subsequent experiments. See an example illustration for an alternative multiplexing strategy below:
Please note: Probe Barcode sequences may be also be included in other kit configurations. For example, BC001 is included in all kit configurations and the sequence is the same in each kit configuration.
When pooling less than 4 or 16 samples, the number of cells targeted per GEM well should be adjusted as explained below:
- When using the Chromium Fixed RNA Kit, Human Transcriptome, 4 rxns x 4 BC PN-1000475 to pool less than 4 samples per well, the total number of cells targeted should not exceed 10,000 cells per Probe Barcode (or 10,000 * number of Probe Barcodes used/per well). For example, if pooling 3 samples hybridized with 3 unique Probe Barcodes, the total number of cells targeted should not exceed 30,000 cells/well (10,000 * 3).
- When using the Chromium Fixed RNA Kit, Human Transcriptome, 4 rxns x 16 BC PN-1000476 to pool less than 16 samples per well, the total number of cells targeted should not exceed 8,000 cells per Probe Barcode (or 8,000 * number of Probe Barcodes used/per well). For example, if pooling 14 samples hybridized with 14 unique Probe Barcodes, the total number of cells targeted should not exceed 112,000 cells/well (8,000 * 14).
The undetected multiplet rate will increase if the recommended number of cells per Probe Barcode is exceeded.
Products: Fixed RNA Profiling, Single Cell Gene Expression Flex