Tprofessional 96

Genotyping of the candidate SNPs LEPREL1:g.139212C>G, KRT3:g.2584C>T as well as the polydactyly-associated polymorphism in LMBR1 (DC-2) was performed using restriction fragment length polymorphisms according to standard protocols [52 (link)]. DC-2 primers were obtained from previous study [18 ]. In addition the missense variants CEP164:g.57380G>T and COL28A1:g.159951T>A were validated using Kompetitive Allele Specific PCR (KASP) genotyping assays (LGC, Middlesex, UK) [53 ]. After the KASP standard thermal cycling touchdown protocol was run on a thermocycler TProfessional 96 (Biometra, Göttingen, Germany) using an annealing temperature of 61 °C (Additional file 12), allelic discrimination was performed on the ABI7300 sequence detection system (Applied Biosystems, Waltham, Massachusetts, USA).

Skin tissues for RNA isolation were available from an SPAID-affected Shar-Pei that was euthanized due to signs of severe renal dysfunction including sickness, diarrhea, fever and swollen joints. In addition skin tissues from an Irish Wolfhound and English Springer Spaniel which were euthanized due to orthopedic problems were used as reference samples. RNA was extracted according to standard protocols for the RNeasy Mini Kit (QIAGEN, MD, USA). Tissues were homogenized in QIAzol Lysis Reagent (QIAGEN) using the Precellys homogenizer (Bertin Technologies, Montigny le Bretonneux, France). The obtained RNA was transcribed into cDNA using the Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Fermentas, Waltham, MA, USA). For Sanger sequencing primers (Additional file 9: Table S9) were designed using Primer3 software (version 0.4.0, http://bioinfo.ut.ee/primer3-0.4.0/) and PCR reactions were run on a thermocycler TProfessional 96 (Biometra, Göttingen, Germany).