Kapa mouse genotyping kit

All animal experiments were performed in accordance with the guidelines for animal experimentation issued by the PNU and were approved by the Institutional Animal Care Committee of Pusan National University (PNU, IACUC approval No. PNU-2021–2937). Wild-type (WT) C57BL/6 J mice were obtained from Hyochang Science (Daegu, Korea). TLR7 knockout (Tlr7^−/−) mice were provided by Dr. Shizou Akira (Osaka University, Suita, Japan). Genotyping was performed using a KAPA Mouse Genotyping Kit (#KK7302, Sigma). To determine the role of TLR7 in chronic kidney disease, male WT mice (8 weeks old, n = 5–6) and male TLR7 knock out (KO) mice (8 weeks old, n = 5–6) were fed a 0.25% adenine diet (AD) for 3 weeks. To determine the effect of the TLR7 inhibitor on kidney injury, a folic acid (FA, dissolved in 0.3 M NaHCO3)-induced kidney fibrosis model was utilized. The TLR7 inhibitor, M5049 (0.3 mg/kg) (dissolved in 0.1 M Na-citrate buffer pH 3.0), was orally administered daily for 2 weeks. For all studies, mice were maintained at 23 ± 2° C with relative humidity of 60 ± 5% under 12 h light/dark cycles. At the end of each study, serum was collected for biochemical analysis, and the harvested kidneys were fixed in neutral buffered formalin for histochemical examination or frozen at − 80 °C for further biochemical experiments.

Ca_v2.3 mutant mice were genotyped by PCR based on the protocol of the KAPA Mouse genotyping kit (Sigma‐Aldrich, Germany). The following primers were used: WT forward 5′‐GGC TGC TCT CCC AGT ATA CT‐3′; WT reverse/KO reverse 5′‐CAG GAA GCA TCA CTG CTT AG‐3′; KO forward 5′‐ATT GCA GTG AGC CAA GAT TGT GCC‐3′. PCR was carried out using the C1000 thermal cycler (Bio‐Rad) with an initial denaturation (94 °C for 3 min) followed by 35 cycles (each cycle containing the following steps: denaturation 94 °C for 15 s, annealing 59 °C for 15 s, extension 72 °C for 15 s) and final extension (72 °C for 1 min). Subsequently, PCR products were separated via agarose gel electrophoresis and detected by ChemiDoc Touch (Bio‐Rad). For details on the procedure and genotyping results see also^{28 ,31 ,38 ,39} . Note that genotyping of all experimental mice was carried out twice per animal (see supplementary tables 4–6) at the post weaning state. Further molecular characterization of the model is provided by Wilson et al. (2000)^{100 (link)}. The reduction/absence of the Ca_v2.3 expression in Ca_v2.3^+/− and Ca_v2.3^−/− mice was further proven by our group using the Western blot approach^{38 ,39} .

Ca_v3.2 mutant mice were genotyped by polymerase chain reaction (PCR) based on the protocol of the KAPA Mouse genotyping kit (Sigma Aldrich, Germany). As described previously, the following primers were used: WT-forward: 5′-ATT CAA GGG CTT CCA CAG GGT A-3′, WT-reverse/ KO-reverse: 5′-CAT CTC AGG GCC TCT GGA CCA C-3′, KO-forward: 5′-GCT AAA GCG CAT GCT CCA GAC TG -3′ (see^{60 (link),62 ,63} ). PCRs were carried out using a C1000 thermal cycler (BioRad, Germany) with initial denaturation (94 °C for 3 min), followed by 35 cycles (denaturation, 94 °C for 15 s; annealing, 61 °C for 15 s; extension 72 °C for 15 s) and final extension (72 °C for 1 min). Finally, PCR products were separated using agarose gel electrophoresis and visualized by ChemiDoc Touch (BioRad, Germany). Examples of our genotyping of Ca_v3.2 mutant mice are provided in detail in^{62 ,63} . Note that genotyping of all experimental animals was carried out twice per animal (see supplementary tables 1–3) at the post weaning state. Further molecular details on the mutant Ca_v3.2 line are also described by Chen et al. (2003)^{60 (link)}. The reduction/absence of the Ca_v3.2 expression in Ca_v3.2^+/− and Ca_v3.2^−/− mice was further proven by our group using the Western blot approach^{62 ,63} .