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.