Enspire workstation

Sera-Mag™ SpeedBeads was prepared according to Faircloth et al. [27 ] and used to clean the PCR products using a Zephyr NGS Workstation (Caliper Lifesciences, Perkin-Elmer) and quantified using a Promega dsDNA Quantifluor System Kit (Ref: E2670, 00002484139) on an Enspire Workstation (Perkin-Elmer). The 5 different PCR products were pooled at approximately 0.4 nM to ensure even coverage during sequencing using Quantifluor dsDNA System (Promega, Madison, WI, USA). The products were normalized to 2 ng/µL using 10 mM Tris-HCl (pH 8.0). Final dilution to 0.2 ng/µL with 10 mM Tris-HCl (pH 8.0) was conducted in preparation for library preparation and final accuracy checks using the Illumina Nextera^XT DNA.

Human orexin-A and orexin-B peptides were purchased from the PEPTIDE Institute (Osaka, Japan). Human wild-type orexin-A cleavage site substrate pLTLGKR-AMC (pyroleucine–threonine–leucine–glutamine–lysine–arginine–aminomethylcoumarin) and human mutant orexin-A cleavage site substrate pLTLGRR-AMC (pyroleucine–threonine–leucine–glutamine–arginine–arginine–aminomethylcoumarin) were synthesized by the PEPTIDE Institute. Recombinant human PCSK1 and PCSK2 were purchased from R & D Systems (Minneapolis, MN). Enzyme activity studies were performed according to the manufacturer’s protocols. In brief, PCSK1 activity was measured as the rate of cleavage of 200 μM synthetic fluorogenic substrates, pLTLGKR-AMC or pLTLGRR-AMC, in 50 μL assay buffer (pH 6.0) including 25 mM MES, 5 mM CaCl₂, 1% Brij-35, and 4 μg/mL recombinant human PCSK1 at 37 °C for 60 min. PCSK2 activity was measured as the rate of cleavage of 100 μM synthetic fluorogenic substrates, pLTLGKR-AMC or pLTLGRR-AMC, in 50 μL assay buffer (pH 5.0) including 50 mM NaOAc, 100 mM NaCl, 0.5% Brij-35, and 0.4 μg/mL recombinant human PCSK2 at 37 °C for 30 min. Cleaved fluorogenic substrate measurements were obtained eight times at 380-nm excitation and 460-nm emission using the endopoint mode in an EnSpire Workstation (PerkinElmer Co., Ltd., Waltham, MA).

PCR products were cleaned using Sera-Mag™ SpeedBeads (Merck KGaA, Darmstadt, Germany) in a Zephyr NGS Workstation (Caliper Lifesciences, Perkin-Elmer; Waltham, MA, USA) and quantified using a Promega dsDNA Quantifluor System Kit (Ref: E2670, 00002484139; Madison, WI, USA) on an Enspire Workstation (Perkin-Elmer; Waltham, MA, USA). The PCR products of all fragments were pooled and normalized to 0.2 ng/µL with 10 mM Tris-HCl (pH 8.0) for library preparation, and final accuracy checks of concentration were performed using the Illumina Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA, USA).