TotalSeq™ – BioLegend’s oligonucleotide conjugated antibodies

 

While traditional flow cytometry allows for rapid, multi-dimensional characterization of cell populations, it lacks the resolution and capacity to perform simultaneous proteomic and genomic analysis on a single-cell scale. BioLegend now offers oligonucleotide-conjugated TotalSeq™ antibodies designed for single-cell proteogenomic analysis via cellular indexing of transcriptomes and epitopes by sequencing, or CITE-Seq. They are also compatible with similar workflows, such as REAP-seq.


In place of fluorophores, each TotalSeq™ clone is conjugated to an oligonucleotide that includes a unique 15 nucleotide (nt) barcode, which converts antigen signal into a readable sequence. Combining antigen detection with single cell RNA sequencing (scRNA-Seq) allows for simultaneous transcriptomic and proteomic analyses in individual cells. Furthermore, since fluorophore and detector availabilities are no longer a factor, a significantly larger number of markers that can be analyzed at once. So far, simultaneous analysis of over 80 surface markers with scRNA-Seq has been reported in the literature.


In addition to unique antigen-specific antibodies, we also carry oligo-conjugated cell hashing reagents, which contain antibodies to ubiquitously expressed cell markers to facilitate sample multiplexing and doublet exclusion. TotalSeq™ antibodies are readily compatible with 10X Genomics scRNA-Seq and Illumina sequencing platforms, but are also adaptable to other droplet-based single-cell workflows with assay optimization.

CITE-seq Workflow Overview:

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General TotalSeq™ antibody schematic:

 

The oligonucleotides conjugated to our TotalSeq™ antibodies have 3 main components: a 5’ PCR handle, followed by a 15 nt barcode unique to the clone, and a 3’ flanking sequence. The 3’ sequence determines compatibility to different scRNA-Seq assays. The example shown above is a schematic for our TotalSeq™-A antibody, which contains a Poly-A stretch on the 3’ end. They are compatible with 10X Genomics 3’ kit (v2), but also with any other system that employs poly-A mRNA capture.

Additional resources:

  • CITE-seq | A collection of resources for performing CITE-seq
  • Simultaneous epitope and transcriptome measurement in single cells: Stoeckius M, et al. 2017. Nat. Methods. 14(9):865-868 PubMed
  • Cell "hashing" with barcoded antibodies enables multiplexing and doublet detection for single cell genomics: Stoeckius M, et al. 2017 bioRxiV Link
  • Multiplexed quantification of proteins and transcripts in single cells: Peterson VM, et al. 2017. Nat. Biotechnol. 35(10):936-939 PubMed