Multi beads shocker instrument

Seeds of cultivated melon accession, Harukei-3 (C. melo var. reticulatus), Natsukei-1 (var. reticulatus), Fuyukei (var. reticulatus), Honey dew (var. inodorus), Spicy (var. cantalupensis), Manshuu (var. makuwa), Ougon-9 (var. makuwa), Awamidori (var. conomon), and wild melon, JSS6 (C. agrestis), were obtained from the Genebank of the National Agriculture and Food Research Organization (NARO) in Japan. Melon plants were grown using the hydroponics method in the greenhouse of the University of Tsukuba in Japan as previously reported^{18 (link)}. For genomic DNA sequencing, apexes of branched shoots were detached from plants and immediately frozen in liquid nitrogen. For tissue-wide RNA-seq study, tissues shown in Fig. 3a and Supplementary Fig. 6 were similarly obtained and frozen in liquid nitrogen. For field RNA-seq study, hole-punched leaf samples were also collected in a weekly manner from July to November as shown in Fig. 6. These samples were crushed to powdery frozen samples using the Multi-beads shocker instrument (Yasui Kikai Corporation, Osaka, Japan) and stored at −80 °C until use.

A total of 204 genes (listed in Supplementary Data S1) from S. coelicolor A3(2) was cloned into the inducible expression vector pTip-QC2^{12 (link)} at the NdeI restriction site (CATATG) in order to arrange the start codon at the same position. For genes whose start codon is other than ATG, the start codon was replaced with ATG. Each of the plasmid vector was introduced into the host strain, R. erythropolis L-88^{27 (link)}. The recombinant strains were precultured in LB liquid media containing 34 µg/ml chloramphenicol at 28 °C for overnight. 2 ml of the preculture was transferred to 20 ml of the same fresh media containing 0.5 µg/ml thiostrepton and cultured with 120 rpm agitation for 16 hours. The cells were harvested and washed twice with 100 mM sodium phosphate buffer. The cell pellets were then resuspended with the same buffer containing 8 M urea and cell disruption was performed by glass beads using the Multi-beads shocker instrument (Yasui Kikai, Japan). The supernatant of the disrupted sample was used as the denatured crude cell extract.