Thermo scientific nanodrop one microvolume uv vis spectrophotometer

Genomic DNA was extracted from the muscle of the pereiopod of all 17 spiny lobsters. DNA was extracted using the AccuPrep^® Genomic DNA Extraction Kit (Bioneer, Daejeon, South Korea), following the manufacturer’s instructions. Concentration and purity of the extracted DNA were measured using a Thermo Scientific™ NanoDrop™ One microvolume UV-Vis spectrophotometer (Thermo Scientific, Wilmington, DE, USA).
A polymerase chain reaction (PCR) was performed to amplify the mitochondrial COI gene region using the HCO1490/LCO2198 universal primers, specially designed for invertebrates (Folmer et al., 1994 (link)). PCR was performed using a 50 µL reaction mixture, consisting of 100 ng genomic DNA, 0.25 µL Taq polymerase (Takara Bio Inc., Shiga, Japan), 5 µL 10X Ex. Taq DNA polymerase buffer (Takara Bio Inc.), 1 µL each of 10 µM forward and reverse primers, and 4 µL (2.5 mM) dNTPs (Takara Bio Inc.). The PCR thermal profile comprised an initial step of 5 min at 94 °C, followed by 30 cycles at 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 45 s, followed by a final extension at 72 °C for 5 min. The amplified PCR products were separated using 1% agarose gel electrophoresis, and target bands were purified using the AccuPrep^® PCR Purification Kit (Bioneer), according to the manufacturer’s instructions.

RNA was isolated from cell lines and human pancreatic islets via Trizol. Briefly, 300 µl of Trizol (Zymo Research, Irvine, CA) was added to each well. After homogenizing, the Trizol reagent was transferred to an Eppendorf tube containing 300 ul of 95% ethanol and thoroughly mixed. The mixture was transferred to a spin column (Zymo Research, Irvine, CA) and RNA isolation was performed following the manufacturer’s protocol. DNA was removed by treatment with DNase (QIAGEN, Germantown, MD) according the manufacturer’s protocol. RNA quality was monitored on 1% agarose gels, and RNA quantification was performed using Thermo Scientific Nanodrop One Microvolume UV-vis spectrophotometer (Thermo Fisher Scientific, Grand Island, NY). qRT-PCR was performed as described (19 (link)). Primer sequences used to determine relative transcript abundance are listed in Table S1.