We do not include any user input for background subtraction to improve reproducibility. To quantify the signal strength of each dot, the background pixel offset and gain should be subtracted. For large arrays with human-derived samples, local variations in offset and gain are significant. Variations were observed even within one cell. As such, we assume that offsets are only homogenous at the dot level. Within each dot, the background offset pixel value is defined as the intensity of the pixel(s) that expresses no fluorescence. As such, this corresponds to the pixel with minimum intensity. Dots with extremely strong signal can fill the entire dot space with fluorescent signal. In these cases, background pixel values would be abnormally high and detected as outliers. Outlier background values will be replaced by local median background value instead.