Kapa hifi mix

ATAC-seq was carried out using the Omni-ATAC protocol [12 (link)]. Visceral adipose tissue (1 g) was placed in cold 1 × homogenization buffer (320 mM sucrose, 0.1 mM EDTA, 0.1% NP40, 5 mM CaCl2, 3 mM Mg(Ac)2, 10 mM Tris pH 7.8, 1 × protease inhibitors (Roche, cOmplete), and 167 μM β-mercaptoethanol, in water), homogenized with Dounce homogenizers, and residual debris was precleared by using 80 um nylon mesh filter. Nuclei were then collected by layering with iodixanol mixture. 50,000 counted nuclei were transferred to a tube containing the transposition mix (25 μl 2 × TD buffer, 2.5 μl transposase (100 nM final), 16.5 μl PBS, 0.5 μl 1% digitonin, 0.5 μl 10% Tween-20, 5 μl water) and mixed by pipetting up and down six times. Transposition reactions were incubated at 37 °C for 30 min in a thermomixer with shaking at 1,000 r.p.m. Reactions were cleaned up with Zymo DNA Clean and Concentrator 5 columns. Library amplification was done with 2 × KAPA HiFi mix (Kapa Biosystems) and 1.25 µM indexed primers using the following PCR conditions: 72 °C for 5 min; 98 °C for 30 s; and 10–11 cycles at 98 °C for 10 s, 63 °C for 30 s, and 72 °C for 1 min.

To construct the mAb64 heavy expression plasmid, PCR was conducted using #64 heavy chain RT-PCR product as template, VH1-LEADER-HindIII as 5’ primer and CH2-IgG-NheI as 3’ primer. In 50μl reaction, the following reagents were added: 25μl of 2X Kapa HiFi mix (KAPA Biosystem), 10μl of #64 heavy chain RT-PCR product, 1µl of 5’ primer (VH1 LEADER-HindIII, 20 nM) and 1μl of 3’ primer (CH2-IgG-NheI, 20 nM), and 13µl of RNA-free water. The PCR reaction condition was 94°C for 2 mins, followed by 30 reaction cycles (94°C for 15 second 63°C for 30 second and 68°C for 100 second. The 1.5 kb heavy chain fragment was extracted from agarose gel. The heavy chain was cloned into mammalian expression vector pJW4303 HindIII and NheI cloning site ⁴⁹ and confirmed by DNA sequencing.

To construct the mAb64 Kappa expression plasmid, PCR was conducted using clone #64 Kappa chain RT-PCR product as template, Vk1/2-Leader-HindIII as 5’ primer and CL2-kappa-BamHI as 3’ primer. In 50 μl reaction, the following reagents were added: 25μl of 2X Kapa HiFi mix (KAPA Biosystem), 10μl of #64 kappa chain RT-PCR product, 1µl of 5’ primer (Vk1/2-Leader-HindIII, 20 nM) and 1μl of 3’ primer (CL2-kappa-BamHI, 20 nM), and 13µl of RNA-free water. The PCR reaction condition was 94°C for 2 mins, followed by 30 reaction cycles (94°C for 15 second, 63°C for 30 second and 68°C for 75 second. The 0.7 kb Kappa chain fragment was extracted from agarose gel. The kappa chain was cloned into expression vector pJW4303 HindIII and BamHI cloning site ⁴⁹ and confirmed by DNA sequencing.