Comprehensive RNA and DNA Isolation Protocol

Total RNA was purified from limb tissues or blood samples using Nucleospin kit II (740955.50; Macherey-Nagel GmbH & Co. KG, Düren, Germany), and cDNA was synthesized using Superscript II reverse transcriptase (18064-014; Invitrogen in Thermo Fisher Scientific, Tokyo, Japan) with oligo(dt) 12–18 primers (18418012, Invitrogen in Thermo Fisher Scientific). In the case of limb tissues (Fig. 1g), the mass of blastema samples was weighed and an equivalent mass of intact limb samples was harvested (see Fig. 1a), and these samples were processed for RNA purification and cDNA synthesis at the same scale. Genomic DNA was purified from blood samples using the Wizard Genomic DNA Purification Kit (A1120; Promega, Madison, WI, USA). Using these DNA samples, PCR was carried out with the KODFX system (KFX-101, Toyobo, Osaka, Japan) on an MJ Mini Gradient Thermal Cycler (PTC-1148; Bio-Rad, Hercules, CA, USA). Primer sets used in this study and cycle numbers for the data in figures are listed in Supplementary Table S4. PCR products were subcloned into Escherichia coli using a TA cloning system (45-0640, TOPO TA Cloning Kit, Dual Promoter; Thermo Fisher Scientific) and then sequenced by Sanger protocols (ABI 3130; Applied Biosystems, in Thermo Fisher Scientific)^{26 (link)}.