FIJI ROI selection and circadian analysis
Image stacks were imported into FIJI (RRID:SCR_002285) (Schindelin et al., 2012) and regions of interest (ROI) were selected using a semi-automated approach (full details in Extended methods; macro and user guide in Supplementary files 4 and 5). Measurements for integrated density were taken for these regions across the stack using the Multi-measure plugin. The time-series was converted to decimalized time relative to hours elapsed from entrained dawn (T0) and was cropped to 150h to standardize between experiments. Period estimation was done using the online platform Biodare2 (https://biodare2.ed.ac.uk) applying the FFT-NLLS algorithm on a data window of 24-120h with expected periods set to 18-34h. Raw data was baseline and amplitude de-trended prior to analysis. Manual inspection of resulting periods ensured that all arrhythmic traces were excluded from further analysis.