Advantage 2 polymerase

Previously reported major deletions at TAS2R43 and -45 loci, ~39k b and ~32 kb in length, respectively, were characterised by multiplex PCR reactions. These were performed using primer sets targeted within, outside, and spanning the deleted regions such that amplifications produced alternate products in deleted and non-deleted alleles (S4 Fig). Gel separation and sequencing of the resulting fragments (Advantage 2 polymerase; Takara Bio Inc.) revealed whether a subject carried zero, one or two copies of each gene.

Plasmids, shown in Supplementary Table S2, were constructed by Gibson Assembly. Gibson assembly primers were designed using NEBuilder version 2.5.2 (https://nebuilder.neb.com/#!/ accessed on 14 March 2021). DNA was amplified from wild-type genomic DNA by PCR using Advantage 2 Polymerase (Cat#639201; Takara Bio USA, Mountain View, CA, USA). Plasmid backbones were double-digested with the necessary restriction enzymes and purified using the Qiagen DNA purification kit (QIAGEN GmbH, Hilden, Germany). DNA inserts were cloned into the digested vectors using the NEBuilder Hifi DNA Assembly Mastermix (E2621S; New England Biolabs, Ipswich, MA, USA) and incubated for 1 h at 50 °C. Newly-assembled plasmids were transformed into GC10 E. coli cells made chemically-competent. Then, 2 μL of the Gibson Assembly mixture was added to the 50 μL GC10 competent cells, placed on ice for 30 min, heat shocked for 30 s at 42 °C, placed on ice for 2 min, and then 950 μL of S.O.C. medium was added. The mixture was incubated at 37 °C for 1 h shaking at 250 rpm and then plated on LB plates with the appropriate antibiotic. Colonies were screened by colony PCR, and positive colonies were cultured in 5 mL of LB + antibiotic overnight. Plasmids were purified using the Wizard Miniprep Kit (Promega Corporation, Madison, WI, USA) and sequenced by Eton Bioscience.

PCR amplification was carried out with custom oligonucleotide primers using Advantage 2 polymerase or Advantage HD polymerase (Takara Bio) on a thermal cycler typically with 30s denaturation at 94 °C, 30s annealing at 50 °C, and 1 min per kb elongation at 62 °C.

DNA from PIRAT ChIRP elutions was amplifed using Advantage 2 polymerase (Takara) and subcloned using the Strataclone TA PCR cloning kit (Agilent), followed by Sanger sequencing (Seqlab GmbH).

Plasmid transfections were performed using Lipofectamine 2000 reagent (Life Technologies) according to the manufacturer’s recommendations. To generate EGFP or 3xFlag-tagged WRAP53β and RNF8 constructs, inserts were amplified by PCR (Advantage 2 polymerase, Clontech) and subcloned into pEGFP-C1 (Clontech) or p3XFlag-CMV10 (Sigma, catalog no. E7658) expression vectors. All primers used for PCR amplifications are listed in Supplemental Table 1.