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Developments in Multimodal Single-Cell Analysis


Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) is a form of multimodal single-cell analysis which uses deoxyribonucleic acid (DNA)-tagged antibodies to measure protein levels whilst analysing the transcriptome.


CITE-seq has provided the data needed to answer important cellular biology questions. However, an issue with CITE-seq is background signalling, which can constitute a sizeable fraction of the sequenced reads complicating analysis and masking biological variations.



It has recently been postulated that staining with the recommended antibody concentrations causes high background. Therefore, it is thought that concentrations of most antibodies in an optimised CITE-seq panel aren’t supposed to reach ‘saturation plateau’ and should instead be within their linear concentration range.



This would involve titrating antibodies to an optimal concentration, which is a lengthy process, and it is yet to be seen whether this would resolve the low signal-to-noise issue entirely. In the experiment detailed below, the BD Rhapsody™ System demonstrated less noise than the 10x Genomics Chromium amongst other benefits.4


Comparing two CITE-seq platforms

This experiment sought to analyse two common workflows for CITE-seq: the BD Rhapsody™ Whole Transcriptome Analysis (WTA) Amplification Kit and AbSeq Reagent Kit and the 10x Genomics Chromium with BioLegend TotalSeq™ reagents. The antibodies selected for this experiment had overlapping clones for both systems.

Firstly, the peripheral blood mononuclear cells were prepared and stained with their respective reagents prior to cell capture. The two protocols were followed to create whole transcriptome and surface protein libraries following cell capture and this data was subsequently analysed on their respective platforms.5


Results of this CITE-seq experiment4


  • BD Rhapsody™ System demonstrated higher specificity with more negative cells near-zero molecule detection outside the target population
  • Only the BD Rhapsody™ System managed to resolve CD28 expressing population of cells 
  • BD Rhapsody™ System had a higher resolution for all clones tested
  • BD Rhapsody™ System demonstrated fewer molecules from the negative population detected, contributing to less noise and allowing positive populations to be resolved


Overall, the BD Rhapsody™ System demonstrated fewer reads from non-target cells and high-resolution CITE-seq surface feature data compared to the 10x Genomics Chromium with BioLegend TotalSeq™ reagents. Read full technical note.


  1. Mulè MP, Martins AJ, Tsang JS. Normalizing and denoising protein expression data from droplet-based single cell profiling. Nat Commun 2022;13.
  2. Xie H, Ding X. The Intriguing Landscape of Single-Cell Protein Analysis. Advanced Science 2022;9.
  3. Buus TB, Herrera A, Ivanova E, et al. Improving oligo-conjugated antibody signal in multimodal single-cell analysis. Elife 2021;10.
  4. BD internal data on file
  5. Protocols used.
  • 10X Genomics Protocol: Chromium Next GEM Single Cell 5’ Reagent Kits v2 (Dual Index) with Feature Barcode technology for Cell Surface Protein & Immune Receptor Mapping (CG000330 Rev D)
  • Rhapsody Protocol: BD Rhapsody™ System mRNA Whole Transcriptome Analysis (WTA) and AbSeq Library Preparation Protocol (23-24118(01)



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