Hifi dna assembly system

Construction of the 5-kb histone repeat designed for this study was performed using the HiFi DNA Assembly system from New England Biolabs. PCR amplification of fragments from existing histone repeats, in addition to in vitro synthesized gblocks (IDT) containing the desired nucleotide changes, was employed for the building blocks of the reaction. Manufacturer’s protocol was followed with slight modification to the incubation time of the reaction. Engineered 5-kb histone repeats were then arrayed to 12 copies in pMultiBac (McKay et al., 2015 ; Meers et al., 2018 ). All histone gene arrays were inserted into the VK33 attP site on chromosome 3L (65B2) (Venken et al., 2006 ).

The mouse UCP1 (UCP1_WT) and the S-nitrosylation resistant (UCP1_SNOR) variants were generated in a pcDNA3.1 backbone (Addgene, V790–20) using cDNA isolated from WT BAT. Site-directed mutagenesis was then done with the NEBuilder HiFi DNA assembly system (E5520S) following the manufacturer’s instructions. Primers used for cloning are listed in the Table S1.
The hTERT A41hBAT-SVF were transfected with either pcDNA-EGFP, UCP1_WT or UCP1_SNOR (2.5 μg/well, pcDNA3.1 backbone) using Lipofectamine 2000 reagent (ThermoFisher Scientific, L3000015) in Opti-MEM medium (ThermoFisher Scientific, 31985–062). At 24–48 hours post-transfection, cells were exposed to isoproterenol (ISO, 1 μM, Sigma, 54750-10-6) in the presence or absence of S-Nitroso-N-acetyl-DL-penicillamine (SNAP, 100 μM, Sigma, N3398) for 2 hours. Cells were then trypsinized, spun at 500xg for 5 minutes at 4°C and assessed for UCP1-dependent oxygen consumption (see below).
HEK293A cells (ATCC, CRL-1573) were transfected with DNA constructs (0.5 μg/well) using Lipofectamine 2000 reagent in Opti-MEM medium. At 48 hours post transfection, the activities of Firefly and Renilla luciferase were measured using the Luciferase Assay System (Promega, E1500) and Renilla Luciferase Assay System (Promega, E2810), separately.