Planar protein microarrays.

Protein microarrays were generated as described previously ^55,56 (https://web.stanford.edu/group/antigenarrays/). In brief, peptides, recombinant proteins, and lysates were diluted at the indicated concentrations in a 1:1 solution of PBS/water and protein printing buffer (ArrayIt) (Supplementary Tables 2–4), aliquoted on 384-well plates, and printed on SuperEpoxy Slides using a NanoPrint LM210 system (ArrayIt). Two independent quadruplicates of each analyte were spotted, and some proteins were used in several versions / preparations from different sources (Supplementary Table 2). Ready-made HuProt Arrays version 3.1 were obtained from CDI labs. Arrays were circumscribed with a hydrophobic marker, blocked overnight at 4 °C in PBS containing 3% FCS and 0.1% Tween-20, and incubated with individual mAbs at a concentration of 1 μg/ml for 1h at 4°C, then washed twice for 20 min in blocking buffer on a rotating shaker. Arrays were then incubated with Cy-3-conjugated secondary goat anti-human IgG (0.8 μg/mL) (Jackson ImmunoResearch) for 1h at 4 °C, then washed twice for 30 min in blocking buffer, twice for 30 min in PBS, and twice for 15 s in water. Arrays were spun dry and scanned with a GenePix 4000B scanner (Molecular Devices). Median pixel intensities for each fluorescent spot were determined with GenePix Pro-3.0 software (Molecular Devices). Z-scores for each row of antigens were calculated for viral antigens, raw intensities were analyzed for GlialCAM arrays. Heatmaps were generated with Morpheus software (The Broad Institute; https://software.broadinstitute.org/morpheus).