CITE-seq staining and sample preparation

To minimize batch effects, for each experiment we processed frozen PBMCs from four different patients at 3 different time points (day 0, day 3, and day 7). After thawing, cells were incubated with FcX block (BioLegend) for 10 min. Cells were then divided into separate aliquots and processed independently for the 3P and 5P protocols.

For the 3P CITE-seq staining protocol, samples were stained simultaneously with the antibody/block pool and a unique hashtag for 30 min. Cells were then washed 3 times in staining buffer (2% BSA, 0.01% Tween in PBS) and filtered using a 40 μm Flowmi filter in PBS and pooled in equal proportions. Cells were loaded into 8 lanes of a 10x Genomics Chip B, at 45,000 cells per lane using the 10x Genomics 3′ v3 GEM kit.

For the 5P ECCITE-seq staining protocol, each sample of cells was first stained with a unique hashtag for 30 min. Cells were then washed 3 times in staining buffer, pooled together, and stained with the antibody panel for 30 min. The pool of cells was then washed 3 times in staining buffer and filtered using a 40μm Flowmi filter in PBS. Cells were loaded into 2 lanes of a 10x Genomics Chip A, at 45000 cells per lane, using the 10x Genomics V(D)J kit (v1).

For both 3P and 5P experiments, first strand cDNA was generated by incubating the emulsions according to the respective 10x Genomics protocol. Emulsions were then broken and nucleic acids recovered Subsequent library preparation steps are detailed in the section below.