Normalization of qPCR gene expression data

For each sample, gene expression of 96 genes was determined from qPCR in a 384-well format as described above. Cycle threshold (Ct) values were normalized using the delta-delta Ct method as previously described [34]. Briefly, genes within each sample were first normalized against endogenous controls by subtracting the sample-specific average Ct value for ACTB, HMBS, and TUBA-1B. For each gene, the resulting delta Ct values were corrected for background from contaminating blood cells by subtracting the average delta Ct value for that gene across the 27 healthy donor reference controls to obtain delta-delta Ct values. Undetermined Ct values were imputed as 40 (Supplementary Table 2). Finally, gene expression was quantified as 2^(-delta-delta Ct) which we refer to as ‘normalized expression.’ All analyses were performed with log₂(normalized expression + 1), with the logarithm serving as a variance-stabilizing transformation and the addition of the constant serving to compress expression values comparable to background. In addition, we set gene expression values to zero when the underlying Ct value was greater than 35 as Ct values in this range are generally not quantifiable.