Our method is dependent on a sufficiently large number publicly available perturbation experiments that activate or inhibit one of the pathways we were looking at. The following conditions needed to be met in order for us to consider an experiment: (1) the compound or factor used for perturbation was one of our curated list of pathway-perturbing agents (for a list, see table S0); (2) the perturbation lasted for less than 24 hours to capture genes that belong to the primary response; (3) there was raw data available for at least two control arrays and one perturbed array; (4) it was a single-channel array; (5) we could process the arrays using available BioConductor packages; (6) the array was not custom-made so we could use standard annotations.
We curated a list of known pathway activators and inhibitors for 11 pathways, where the interaction between each compound and pathway is well established in literature. We then used those as query terms for public perturbation experiments in the ArrayExpress database 27 and included a total of 223 submissions and 573 experiments in our data set, where each experiment is a distinct comparison between basal and perturbed arrays. If there were multiple time points, different cells, different concentrations, or different perturbing agents within a single database submission, they were considered as different experiments.