The standard 10x Genomics recommendation for the library preparation protocol is that the maximum number of cells per library is 10,000. Generally, most users will assay 10,000 cells from one sample. Occasionally users may have a set of samples that individually have fewer than 10,000 cells each, but that can add up to 10,000 cells (so, for example, sample A has 2000 cells, sample B has 3000 cells, and sample C has 5000 cells). It is possible to combine these samples into one library by tagging the cells in each sample with a unique identifier. This is referred to as a hashtag. Oligonucleotide-antibody conjugates are available for this from external vendors, for example, BioLegend (TotalSeq A and B). Alternatively, a lipid-based oligonucleotide tagging system is available from 10x Genomics. Details on each of these are available from the appropriate vendor. Cells that have hashtags are identified informatically after alignment to the reference genome.

The IGL does not do hashtagging. It is the responsibility of each user to select their tagging method, to treat their cells, and to mix them prior to delivery to the IGL for processing. The IGL is able to complete the libraries for these protocols.

To process the samples appropriately, users need to add the oligonucleotide sequence used for each sample to the sample submission form they deliver to the IGL.

Clients can do some basic proteomics work with their single-cell gene expression studies by using antibody-oligonucleotide conjugates. Unlike work with hashtags which use antibodies to universal cell surface proteins, the antibodies used for CITE-Seq target specific cell-surface proteins of interest. Differential analysis of the oligonucleotides after cluster analysis of the sequence data can provide insight into translation and recycling of the targeted proteins. Multiple CITE-Seq oligo-antibody conjugates can be used in a single experiment and CITE-Seq can be combined with hashtags as needed.