The remaining isolated RNA utilized for RNA-sequencing was used to confirm transcriptomics findings using RT-qPCR. RNA concentrations were determined by Nanodrop (ThermoFisher) and complementary DNA (cDNA) was generated from 1μg of total RNA using the iScript Reverse Transcription Supermix for RT-qPCR. (BioRad, Cat No: 1708840) The qPCR C5ar2 primer sequences were purchased from Integrated DNA Technologies (IDT) and designed as described previously (Miyabe et al. 2019) (Forward: 5′-ACCACCAGCGAGTATTATGACT-3′ and Reverse: 5′-GCAGGACTATCAGGTAGACATCA-3′) while Pla2g3 (Cat No: qMmuCIP0028420), Kcnj15 (Cat No: qMmuCEP0042967), and Ubc (Cat No: qMmuCIP0034811) were purchased from BioRad. qPCR was conducted by using the SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad, Hercules, CA; Cat No: 1725270) on an CFX Connect Real-Time PCR Detection System (Bio-Rad). The relative amount of each transcript was determined via normalization across all samples to the endogenous control, GAPDH. cDNA samples from each individual animal were run in duplicate. To quantify the relative expression levels of the genes for each mouse genotype, we calculated the difference (ΔCt) between the cycle threshold of each gene and the housekeeping gene, GAPDH. From these data, the ΔΔCt ([ΔCtGene(HAP)–ΔCtGene(LAP)]) was
