Improving oligo-conjugated antibody signal in multimodal single-cell analysis

Abstract

Simultaneous measurement of surface proteins and gene expression within single cells using oligo-conjugated antibodies offers high-resolution snapshots of complex cell populations. Signal from oligo-conjugated antibodies is quantified by high-throughput sequencing and is highly scalable and sensitive. We investigated the response of oligo-conjugated antibodies towards four variables: concentration, staining volume, cell number at staining, and tissue. We find that staining with recommended antibody concentrations causes unnecessarily high background and amount of antibody used can be drastically reduced without loss of biological information. Reducing staining volume only affects antibodies targeting abundant epitopes used at low concentrations and is counteracted by reducing cell numbers. Adjusting concentrations increases signal, lowers background, and reduces costs. Background signal can account for a major fraction of total sequencing and is primarily derived from antibodies used at high concentrations. This study provides new insight into titration response and background of oligo-conjugated antibodies and offers concrete guidelines to improve such panels.

Normalizing and denoising protein expression data from droplet-based single cell profiling

“…Thus, protein counts in empty droplets, which are available in all single-cell droplet experiments, provide a direct estimate of the ambient background due to free antibody capture for each protein. Consistent with our findings on ambient antibody capture as the major source of background noise in CITE-seq data, a recent study reporting CITE-seq antibody titration experiments across a` wide concentration range demonstrated that background noise increased with the antibody staining concentration, with some antibodies at or above 2.5 µg/mL having even more cumulative UMIs in the empty droplets compared to cells 28 . Our observation thus motivated the first step of our method to remove protein-specific technical noise: transforming counts of each protein in cell-containing droplets by subtracting the mean and dividing by the standard deviation of that same protein across empty droplets (see the “Methods” section).…”

Integration of single-cell RNA and protein data identifies novel clinically-relevant lymphocyte phenotypes in breast cancers

“…Furthermore, some markers such as naive T cell marker CD44 are ubiquitously expressed at a high density across cell lineages and therefore challenging to use with cell-type unbiased CITE-Seq (Saturates CITE-Seq cDNA library) (Buus et al, 2021). Conversely markers commonly employed to assess T cell activation and/or migration state can be degraded by tumour dissociation (Autengruber et al, 2012).…”

Acquisition of murine splenic myeloid cells for protein and gene expression profiling by advanced flow cytometry and CITE-seq

“…CITE-seq is a rather young technology and resources to optimized protocols currently remain sparse. Useful resources regarding determining the optimal concentration of oligo-tagged antibodies can be found here ( Mair et al., 2020 ; Buus et al., 2021 ).…”