Nb100 125

Animals’ blood samples were first collected by cardiac puncture after anesthesia, and then immediately sacrificed. Serum samples were isolated from the blood samples after centrifugation (1000 g, 10 min, 4℃) and stored at −80℃ for later analysis. One thyroid gland lobe was stored at −80℃ having been frozen for 10 min in liquid nitrogen. Another thyroid gland lobe was placed in 4% PFA for 12 h, and underwent gradient dehydration in 15%, 30% sucrose for 2 days. Tissue sections were cut to 2 μm thickness. Primary antibodies against BMAL1 (NB100-2288, dilution 1:500; Novus, USA) and PER2 (NB100-125, dilution 1:250; Novus) and HRP-conjugated secondary antibodies (1:2000) supplied in a DAB kit (CW2069S, CWBIO, Beijing, China) were used for immunostaining of thyroid sections. To validate the specificity of the antibodies used for immunocytochemistry, we performed primary controls (+primary antibody/−secondary antibody) and secondary controls (−primary antibody/+secondary antibody), both of which showed no positive labeling. Immunohistochemistry images were taken with Leica Aperrio Versa 8 microscope (Leica), details of quantification by IHC Profiler (20 (link)) were listed as Supplementary Data 1.

DNA isolation for ChIP-qPCR analysis was performed using the protocol by van den Boogaard et al. (74 (link)). Briefly, several hearts (four to five hearts per sample) were isolated from P7 WT C57BL/6 mice and fixed in 1% paraformaldehyde followed by quenching with glycine. The heart tissue was subsequently homogenized and lysed using 1% SDS lysis buffer and protease inhibitor cocktail (Roche). DNA was fragmented using 20 cycles of 30 s “on” and then 30 s “off” of 50% power sonication, and DNA shearing efficiency was confirmed by DNA electrophoresis. Samples were subsequently incubated with protein G magnetic beads for 1 hour. Samples (2.5%) were kept as input (reference sample). Per2 ChIP-grade antibody (Novus, NB100-125) was added, and samples were incubated at 4°C overnight. Protein G magnetic beads were incubated for 1 hour and then pulled down. Chromatin was eluted from the beads and then uncrosslinked by incubating overnight at 65°C. DNA was purified using phenol-chloroform following ribonuclease A (RNase A) and proteinase K treatment (Thermo Fisher Scientific). DNA concentrations were calculated using the DNA Qubit 4 Fluorometer (Thermo Fisher Scientific). qPCR was performed using SYBR Select qPCR Mix (Thermo Fisher Scientific) with specific primers. Fold enrichment of DNA fragments compared to the input samples was calculated.