2.1. Liver-Specific Conditional Knockout Mouse Model
To generate liver-specific Tip60 knockout mice (mutant), Tip60 floxed mice (10-12 week old male) with loxP sites flanking exons 1 and 9 of the Tip60 gene  were crossed to a SACre driver mouse line resulting in Cre-mediated deletion of Tip60 in the liver . Mice were previously backcrossed to a C57BL/6N background for at least 10 generations. To delete Tip60, 10-12 week-old male mice (Tip60fl/- ; SA+/Cre-ERT2) were injected daily with tamoxifen (10mg/ml stock solution, Sigma, St. Louis, MO, USA) in corn oil for five consecutive days. The control group (Tip60fl/fl;SA+/+), in which corn oil were injected. Genotyping was performed with gene-specific primers [21, 26]. Liver and kidney tissues were collected five days after the last injection. Genomic DNA was isolated from both tissues and analyzed by southern blot.
2.2. Southern Blot Analysis
Genomic DNA from liver and kidney tissues was isolated with the DNeasy Tissue kit (Qiagen Inc., Valencia, CA, USA) and digested with BamHI (NEB, Ipswich, MA, USA). DNA was separated on a 0.6% agarose gel and transferred onto Hybond-XL positive charged nylon membrane (GE Healthcare/Amersham Biosciences, Sweden). The membranes were hybridized with a 32P-dCTP labeled radioactive double-stranded DNA probe was prepared by random priming using an appropriate commercial kit according to manufacturer’s instructions (Amersham Rediprime™ II DNA Labeling System, GE Healthcare) and purified with the illustra ProbeQuant™ G-50 Micro Columns (GE Healthcare). Hybridization of the radioactive probe (100 µl) to the membrane was performed at 65°C overnight in the presence of a hybridization buffer. Membranes were washed with 2xSSC/0.1% SDS, 1xSSC/0.1% SDS, and 0.1xSSC/0.1% SDS, at 60°C until the excess label was removed and exposed to a sensitive X-ray film (Kodak X-Omat 1000,1000A ve 1000J Processors).
2.3. Experimental Animals, Feeding, and Zeitgeber Time
At least 3 weeks prior any experiment, all mice were singly housed with food and water ad libitum under a 12‐hour‐light/12‐hour‐dark cycle (350 lux). Throughout this study, time is indicated using zeitgeber time (ZT) as the indicator for the phase of the rhythm, wherein ZT0 refers to the time that lights went on (06:00), and ZT12 refers to the time that the lights went off (18:00). ZT4, ZT8, ZT16, and ZT20 in this study are equivalent to 10:00, 14:00, 22:00, and 02:00 respectively . Artificial light was provided daily from ZT0 (06:00), with temperature (24 ± 1) °C, and humidity (55 ± 5%) kept constant . In the first set of experiments, 10–12‐week‐old male C57BL/6N mice were used and split up into six groups corresponding to the six chosen timepoints (ZT0, ZT4, ZT8, ZT12, ZT16, and ZT20). For the second set of experiments, we used 10–12‐week‐old male mutant mice (Tip60fl/- ; SA+/Cre-ERT2) and their respective control littermates .
2.4. RNA Extraction and First Strand cDNA Synthesis
Total RNA isolation from approximately 50 mg mice liver tissues was performed using the RNeasy Lipid Tissue Mini Kit (Qiagen-74804) following the manufacturer’s instructions. Concentrations and purities of RNAs were measured by spectrophotometer. (Thermo Scientific, Multiskan GO, USA), RNA quality was checked by agarose gel electrophoresis and stored at −80°C until use. Total RNA was converted into first strand cDNA using SuperScript III First-Strand cDNA kit system (Invitrogen, California, USA), utilizing random hexamers, according to the manufacturer's protocol. The resulting cDNAs were diluted to 100 ng/μL with nuclease-free water and stored at −20 °C .
2.5. Primers and Probes Design
Primer3 software (v. 0.4.0) (http://bioinfo.ut.ee/primer3-0.4.0/) was used for gene-specific primers and probes design that met the following criteria; amplicon size 75-200 bp, ≤3 G or C repetitions, GC content 50-65%, ≤4 base repetitions, melting temperature (Tm) 60°C. Primers and probes were verified with Blast Tool (NCBI) to confirm its specificity for the desired target. Then they were synthetized and purchased from Methabion International (Martinsried, Germany). Gene symbols and GenBank ID numbers are; Ca1 (Gene ID: 12346), Ca3 (Gene ID: 12350), Ca7 (Gene ID: 12354), and Actb (Gene ID: 11461). Since housekeeping genes were not affected by any of treatments, β-actin was used as reference gene. The sequences of specific primers of all genes were shown in Table 1.
2.6. Quantitative Real‐Time PCR
To determine expression levels of Ca 1, Ca 3, and Ca 7 genes in different circadien points, Real-time PCR (qPCR) was carried out on Rotor-Gene Q instrument (QIAGEN, Inc., Hilden, Germany). Beta-actin was selected as reference control gene. The qPCR reactions were carried out with 2 µL of cDNA (final concentration is 0.02ng), 4 pmol of TaqMan probe, 8 pmol of forward and reverse primers and 10 µL FastStart TaqMan Probe Master Mix (Roche Diagnostics GmbHCorp, Mannheim, Germany) in a final volume of 20 µL. Optimal cycling conditions were 50 °C for 2 min, 95 °C for 10 min, 45 cycles of 95 °C for 15s, and annealing and extension at 60 °C for 1 min . The expression results were analyzed using the ΔCT method .
2.7. Statistical Analysis
Each group contained three animals, and all measurements were triplicated for each animal. Statistical analysis was performed for each experiment using one‐way and two way analysis of variance (ANOVA) with Tukey's and Bonferroni's multiple comparisons test using the Prism software 7.0 (GraphPad Software, San Diego, CA). A value of P < .05 was considered to indicate a statistically significant difference (*P < .05, **P < .01, ***P < .001, **** P < .0001).