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. In this study, 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 cause unnecessarily high background and that concentrations can be drastically reduced without loss of biological information. Reducing 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 the total sequencing and is primarily derived from antibodies used at high concentrations. Together, this study provides new insight into the titration response and background of oligo-conjugated antibodies and offers concrete guidelines on how such panels can be improved.
Impact statement Oligo-conjugated antibodies are a powerful tool but require thorough optimization to reduce background signal, increase sensitivity, and reduce sequencing costs.
Competing Interest Statement
PS is co-inventor of a patent related to the single cell technology utilized in this study (US provisional patent application 62/515.180).
Footnotes
In the revised manuscript, we have done our best to address all the points raised by the preprint peer-reviewers. We have conducted an additional CITE-seq experiment comparing the signal and background from pre-titration concentrations with our suggested concentrations for all 52 antibodies as well as using our recommended reduced staining volume and cell numbers. This experiment shows marked improvements in signal and sequencing balance, as well as reduced background across the panel. In addition to the improved signal-to-noise, we estimate that the antibody costs of our suggested approach is ~34 fold cheaper than using vendor recommendations and requires less sequencing depth to acquire same signal. Prompted by the suggestions from our reviewers we have also updated the figures to improve clarity, show cell type annotations and included a guide to the high-information-content titration plots presented in the manuscript.
