SPARC single-cell protein data

Protein data measurements were carried out using PEA and quantitative values on protein concentrations were measures using qPCR. Following the completion of the qPCR run we visually inspected the amplification curves. Samples showing evidence of failed or poor amplification reactions were excluded from further analysis. Next, the raw Cq data (log 2 scale) was exported and processed. First, samples were excluded if no signal was detected in any of control assays (extension control, incubation control, or detection control), or if the signal in any of the controls was greater or less than 2 standard deviations (SD) of the mean value across all samples measured on a 96.96 IFC Biomark chip. Next, the remaining Cq values were normalized for intra-plate variation with the extension control (Cqassay—CqExtCtl) yielding dCq values. Then, for each assay, the dCq values were subtracted from the negative control computed as the lysis buffer mean + 2 × SD. This ensures that observed signals for each assay in the presence of a cell are at least 2 SDs away from any signals observed in the absence of any antigen. Resulting values below zero were set to zero and the signal was deemed undetected. The cumulative protein sum was calculated by summing across all proteins measured (n = 92) per cell.