a, 110,592 clones from a 6-day tail blastema library were arrayed on 288 × 384-well plates. b, One 384-well plate was pooled into one conical tube and called a ‘pool’. In total, 288 pools were prepared from the library. c, Twenty-four pools were combined in one conical tube and called a ‘superpool’ (SP) containing 9,216 clones. In total 12 superpools were prepared. d, Bacteria of each superpool was cultured and plasmid was prepared. e, The superpool plasmids were transfected into HEK293 cells. f, Individual supernatants were tested on A1 myotubes for cell cycle re-entry activity (myotube assay) (see Fig. 1b). Positive superpools were successively subfractionated and the assay process was repeated back from the positive superpool (first screen) to come to a single clone (fourth screen) (a–c, right) (see Extended Data Fig. 2a–c).