Fig 1: Discrete and specific differences in the methylation profiles of Afh/Afh SCN vs +/+ animals. a Schematic representation of the Opn4 locus highlighting the CGI region (upper panel) at the beginning of the transcription start site (TSS, black arrow), as analyzed by pyrosequencing. Box plot of methylation values for each biological replicate averaged across all of the CpGs in the pyrosequencing amplicon (lower panel); the red lines represent the median, and the whiskers represent the 5th and 95th percentiles. b Pyrosequencing of Opn4 CGI methylation levels in Afh/Afh and +/+ mice at the two different time points, ZT0 and ZT12. c Principal component analysis of CGIs methylation vs time of day for +/+ and Afh/Afh animals after RRBS sequencing. d Logistic regression of the RRBS data identified 31 differentially methylated CpG islands (CGIs) (with a difference greater than 10%). Quantitation of methylation levels was performed with the SeqMonk software suite considering CGIs with a minimum count of 5 per position and at least 3 observed positions for each CGI across the dataset. The coloured scale on the right indicates the hypermethylated CGIs (in green) and the hypomethylated CGIs (in red). e Relative expression of a subset of genes with differentially methylated CGIs identified by the RRBS analysis at the two different time points, ZT0 and ZT12, normalized to the levels of multiple housekeeping genes. The bars represent the average values of three different experiments ± SEM. * p < 0.05, ** p < 0.01, two-way ANOVA plus Bonferroni post-test (b), Student’s t test (a), two-way ANOVA with Tukey’s post hoc test (e). a–b n = 5 for Afh/Afh and n = 6 for +/+ . c n = 6 for each genotype. d n = 3 for Afh/Afh and n = 3 for +/+ at each time point. e Minimum n = 7 per genotype at each time point