Lighercycler 480

Retrotranscription to first strand cDNA was performed using RevertAid H Minus First Strand cDNA Synthesis kit (Themo Fisher Scientific ref: K1632). Briefly, 2000 ng of total RNA was used for cDNA synthesis following manufacturer’s instructions. 5 ng of original RNA was used to perform fast qPCR using GoTaq qPCR Master Mix (Promega ref: A6002) in a LigherCycler 480 (Roche) with the manufacturer’s protocol (Supp. Table 3). Primers were designed using OligoPerfect design (Thermo Fisher Scientific) and validated using in silico PCR (UCSC genome Browser) and Ensembl BLAST (Ensemble.org). Primers were purchased from Sigma-Aldrich (Supp. Table 4) and used at 0.5 μM final concentration. Finally, primer efficiency was tested using serial dilutions from a mixed pool of cDNAs from all the samples. All primers used showed an efficiency of 1.9 or above (Supp. Table 4).

NRK cells were transfected with scrambled or Dvl1 shRNA clones as described above using electroporation and Nucleofector (Lonza). After 24 h, cells were washed in cold PBS and homogenized using Trizol Reagent (Life Technologies). Total RNA was then extracted using Direct-zol columns (ZymoResearch). RNA concentration was quantified using a NanoDrop ND-100 (Thermo Scientific). Up to 2000 ng of RNA were used for cDNA synthesis using RevertAid H Minus First Strand cDNA Synthesis kit (Themo Fisher Scientific). Five nanogram of original RNA was used to perform fast qPCR using GoTaq qPCR Master Mix (Promega) in a LigherCycler^® 480 (Roche). Primers for Dvl1 and three housekeeping genes (Gapdh, Actb and Rps18) were designed using OligoPerfect design (Thermo Fisher Scientific) and validated using in silico PCR (UCSC genome Browser) and Ensembl BLAST¹. Primers (Dvl1 Fw: 5′-GCTGAAGCATGGTTTCCTGC-3′; Dvl1 Rv: 5′-GTTGAGGTTCAGGGATGCGA-3′; Actb Fw 5′-GGCTCCTAGCACCATGAAGA-3′; Actb Rv: 5′- CTGGAA GGTGGACAGTGAGG-3′; Gapdh Fw 5′-AGACAGCCGCATC TTCTTGT-3′; Gapdh Rv: 5′-CTTGCCGTGGGTAGAGTCAT-3′; Rps18 Fw 5′-CTTCCACAGGAGGCCTACAC-3′; Rps18 Rv: 5′-GTACTCGCAGGATGTGCTGA-3′) were used at 0.5 μM (Sigma Aldrich).