Yeast One-Hybrid (Y1H) screen

By using human substantia nigra total RNA and Matchmaker One-Hybrid Library Screening Kit (Takara Bio USA Inc., Mountain View, CA, USA, Cat# 636560 and 630304), we screened for transcription factors in the following six steps per manufacturer’s instruction: (a) constructing and testing the bait which was prepared by annealing two complementary 40 base DNA oligonucleotides harboring dinucleotide polymorphism in Intron 1 allele B(DNPiB) in the middle and carrying EcoR I and Mlu I at each end for inserting to reporter pHIS2.1 (see Supplementary Table 1 for all nucleotides used in this study); (b) generation of the cDNA for the library including first strand cDNA synthesis, amplifying cDNA using Long Distance PCR (LD-PCR), and purifying ds cDNA with CHROMA SPIN TE-400 Columns; (c) library screening; (d) confirmation of positive interactions and rescue of the prey plasmid by re-streaking and yeast colony PCR to eliminate duplicates, rescuing and isolation of library plasmids responsible for activation of reporters; (e) yeast transformation followed by streaking single candidate colony on new 100mM 3-AT, TDO (Triple Dropout Medium: SD/–His/–Leu/–Trp) plate for selection; and (f) screening by PCR of selected colonies and DNA re-sequencing.