When cells stained by these antibodies are lysed, the mRNA is captured by the bead and the ADT is also captured in an identical fashion. The captured mRNA can be processed into a scRNA-seq library. Since the ADTs are short, they can be isolated from the cDNA by size using magnetic beads. ADTs are then processed into an independent ADT library because they have an incorporated PCR handle. The ADT libraries can be sequenced independently, but they are usually pooled with their respective cDNA library. Typically, the ADT library represents 5-10% of the pooled libraries and is sequenced at a read depth of 5,000-10,000 reads/cell (depending on how many ADTs are used). Our TotalSeq™-A line of reagents was designed with a poly(A) sequence, making it platform agnostic and allowing it to work with most, if not all, poly(dT) capture methods.
 

Though they use different capture sequences, there are no significant differences in identifying positive or negative populations when using either poly(dT) capture with TotalSeq™-A or feature barcode capture using TotalSeq™-B reagents. While there may be a slightly lower capture of mRNA when using TotalSeq™-A since there is competition for binding sites, this difference is negligible. The rate of capture between TotalSeq™-A and -B are inherently different, but since the libraries are processed independently, they are comparable bioinformatically. While TotalSeq™-A and B can be used simultaneously, it can be difficult to bioinformatically deconvolute the data, and therefore, is not advised.

TotalSeq™-C functions similarly to TotalSeq™-A; however, TotalSeq™-C is designed to work with 10x Genomics’ 5’ Immune Profiling kits. Since this platform utilizes a 5’ capture, the beads do not contain a poly(dT) capture sequence. Instead, an anti-template switching oligo (TSO) sequence is used. TotalSeq™-C antibodies contain an oligonucleotide sequence that has been altered to suit this different capture method.

In addition to new single-cell platforms, CITE-seq has been expanded to include additional multiomics parameters. For example, ATAC with Select Antigen Profiling by sequencing (ASAP-seq) brings CITE-seq technology to single-cell ATAC sequencing (scATAC-seq) to assess genome-wide chromatin accessibility. scATAC-seq beads have a unique capture sequence from those discussed above. These capture sequences have a cell barcode but no UMI, since there are only two copies of a gene in a cell. Instead of designing new oligo tags for the new capture sequence like TotalSeq™-C, a bridging oligo is used (Fig. 3). This bridging oligo is able to bind to the capture sequence on the bead as well as the capture sequence of the ADT. The bridging oligo also contains a unique bridging identifier (UBI) to add the resolution and controlling for PCR duplication similar to a UMI.  

Cell Hashing

Another useful technique using oligonucleotide-conjugated antibodies is cell hashing. Using antibodies against markers that are ubiquitously expressed across different cell types, each sample can be tagged with a different oligonucleotide barcode. Our TotalSeq™-A, -B, and -C formats contain hashtag antibodies available for human and mouse specificities. TotalSeq™ hashtag reagents are a mix of two clones. Human hashtag reagents target CD298 and β2 microglobulin, and mouse hashtag reagents bind CD45 and MHC class I (a, b, d, j, k, s, and u haplotypes). This allows for the tagging of separate samples, which can then be pooled into an independent single-cell run (Fig. 4).

The labeled cells are identified by their unique hashtag and assigned to their sample group. This has the added benefit of identification of doublets/mutiplets. If a data point has more than one hashtag, it is very likely to be two or more cells that were captured together. Unfortunately, this doesn’t account for cells with the same hashtag being captured together. Still, being able to identify mutiplets opens the possibility of “super loading” and expanding the number of loaded cells. Without hashtagging, this is difficult as an increase in input cells in a single-cell sequencing experiment increases the likelihood of doublet formation. This limits the number of cells that can be loaded, as doublets/multiplets would begin to occupy too many data points. By hashtagging samples and identifying multiplets for exclusion, many more cells can be super loaded compared to conventional methods. It should be noted that super loading does have diminishing returns as the doublet rate increases.

Nuclear hashtags are also available in our TotalSeq™-A format. These bind to nuclear pore complex proteins and retain all of the cell hashing benefits for nuclei in single-nucleus RNA-seq (snRNA-seq) experiments.

 

BEN-seq

TotalSeq™ antibodies are not only useful in single-cell sequencing experiments, but also in bulk RNA-seq. In collaboration with Illumina, we developed a Bulk Epitope and Nucleic acid sequencing (BEN-seq) workflow to leverage the ability to measure surface proteins by sequencing. Samples are processed in a similar way to CITE-seq, except that the readout provides a holistic view of surface protein expression and cDNA from a population of cells. BEN-seq can easily be adapted to work with typical bulk RNA-seq workflows and reagents. Learn out more about BEN-seq in our application note.

Multiomics has transformed traditional sequencing experiments and can provide unparalleled protein and genetic analysis in a single experiment. Explore our TotalSeq™ reagents and see how they can be incorporated into your workflow. And if you can’t find the reagents you’re looking for, contact us to discuss custom oligo-conjugated antibodies, hashtags, or antibody cocktails for your experiment.  

References:

1. Stoeckius et al. Simultaneous epitope and transcriptome measurement in single cells. Nat Methods. 2017 Sep; 14(9): 865-868.
2. Peterson et al. Multiplexed quantification of proteins and transcripts in single cells. Nat Biotechnol. 2017 Oct; 35(10): 936-939.