Brain matrix

After 1 hr light pulse, animals were killed under dim light by cervical dislocation. To prevent any photic stimulation to the SCN, eyes were immediately removed. Removed brains were placed into a brain matrix (Kent Scientific, Torrington CT, US). Two skin graft blades (Kent Scientific, Torrington CT, US) one positioned at Bregma −0.10 mm and the second at Bregma −1.10 caudal from the first, were used to cut a 1 mm thick brain slice. SCN punches were collected from the brain slices by using a sample corer (1 mm internal diameter, Fine Science Tools GmbH, Heidelberg, Germany). SCN punches were stored at −80°C prior to RNA extraction.

WT C57BL/6J mice (males, aged between 8 and 12 wk) were used for SCN tissue collection as described by Jagannath et al. (2013) (link) at six distinct time points, starting from ZT3, at every 4 h, with lights on at 7 am (ZT0) and lights off at 7 pm (ZT12). We also collected cortical punches at ZT3 and ZT15 to compare and establish SCN-enriched chromatin modifications. An approximately 1-mm-thick mouse brain slice was sectioned between Bregma −0.1 and −1.0 mm using a brain matrix (Kent Scientific) and sterilized razor blades. The dissected mouse brain slice was placed on a cold block and promptly checked under a light microscope for the SCN (Bregma −0.3 and −0.8 mm, rostral to caudal) using cell-density contrast. Thereafter, the SCN (and cortex) was collected using a sample corer (1-mm internal diameter, Fine Science Tools) from the brain slice, flash-frozen on dry ice, and stored at −80°C. For histone ChIP, two separate biological replicates per time point per tissue type were collected, where each biological replicate comprised three to four individual SCN or cortical punches. For SCN-bulk RNA-seq, four biological replicates per time point were collected. Likewise, each biological replicate constituted three to four individual SCN samples.