Optimizing Experimental Design for High-Parameter Protein Analysis Using BD AbSeq Technology

High-throughput single cell RNA sequencing (RNA-seq) has recently emerged as a powerful tool for profiling complex cell populations. While traditional single cell RNA-seq captures information about transcript expression, recent technological advances using oligo-conjugated antibodies enable simultaneous detection of proteins alongside mRNA in high-throughput sequencing. This multiomic technology enables high parameter protein analysis that can simultaneously discriminate 100 protein markers. However, experiment workflow time and cost can escalate quickly with high plexy assays. To address these issues we investigated a number of methods to decrease cost and assay time while maintaining data quality using the BD® AbSeq on the BD Rhapsody™ Single-Cell Analysis sequencing system. To begin, we investigated the performance of 20 core proteins when used in a 20plex, 40plex, 60plex, 80plex, or 100plex in model cells. Results from 3 experiments showed that performance was maintained as plexy increased, however rigorous experimental approaches must be followed to minimize impact on specificity or sensitivity. After establishing performance of this 20plex we investigated pre-cocktailing of antibodies as a method to shorten the day-of-experiment workflow, and mixing of oligo-labeled and unlabeled antibodies to high-expressing proteins (“signal muting”) as a way to decrease sequencing costs. Results showed a decrease in specificity as soon as 24 hours after the cocktailing of antibodies and increasing non-specific binding with longer cocktail storage times. On the other hand, signal muting was effective at decreasing sequencing requirements by up to 70%, without impacting data for high expressors. Together these results can be used to aid in optimizing experimental design when working with high-plex AbSeq protein panels, to better harness the information gained from multiomic single cell sequencing data. For Research Use Only. Not for use in diagnostic or therapeutic procedures.BD, the BD Logo, and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2019 BD. All rights reserved. Citation Format: Punya Narayan, Hye-Won Song, Margaret Nakamoto, Elisabeth Walczak, Katherine Lazaruk, Stefanie Mortimer. Optimizing experimental design for high-parameter protein analysis using BD AbSeq technology [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1348.