Rnaeasy micro

qPCR analysis was performed with the QuantStudio 6 Flex Real‐Time PCR System (Applied Biosystems, CA). Total RNA was extracted with RNAeasy Micro (cells), Trizol, and/or Mini Kits (tissues) (Qiagen, CA). One thousand nanogram total RNA from each of the pooled PVC cells/tissues or atrial tissue samples was reverse transcribed with SuperScriptTM First Strand Synthesis System for RT‐PCR (Invitrogen, CA). Real‐time PCR was performed in triplicates for every sample using primers listed in Table 1. Using SYBR Green/ROX probe (Thermo Fisher, MA), averaged C_t values of each qPCR reaction from the target gene were normalized with the average C_t values of the housekeeping gene 18S, which ran in the same reaction plate to obtain the ∆C_t value (Barajas‐Martinez et al. 2017; Goodrow et al. 2017). Expression was normalized from ∆C_t values for each gene against reference housekeeping gene 18S, using the formula 2^−∆∆Ct (1 × 10⁶) (Livak and Schmittgen 2001):

3-10k cells were FACS sorted into buffer RLT with β-mercaptoethanol and total RNA prepared using RNAeasy Micro (Qiagen) with on column DNase I treatment. Stranded RNAseq libraries were prepared using the TotalScript RNAseq kit (Epicenter). Libraries were sequenced paired-end (2x50 cycles) on the Illumina platform (Illumina). Reads were mapped using STAR (v 2.5.2b) (32 (link)), strand-specific reads in exons quantified using HOMER (33 (link)) and significant changes identified using EdgeR (34 (link)). Only genes with ≥30 reads in at least two samples were considered in the analysis. Data was log transformed and quantile normalized for display. PCA and visualization were performed using R (v3.3.3). GO term analysis was done using Metascape (35 (link)) with standard settings.

The gene expression of endothelial nitric oxide synthase (eNOS), protein kinase RNA-like ER kinase (PERK), activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1), CCAAT-enhancer-binding protein homologous protein (CHOP), 78 kDa glucose-regulated protein (GRP-78), protein phosphatase 1 regulatory subunit 15A (PPP1R15A), and the unspliced and spliced forms of X-box binding protein 1 (XBP1) mRNA were measured by real-time quantitative PCR. Total RNA was extracted from HUVECs using a kit (RNAeasy micro, QIAGEN, Tokyo, Japan). cDNA was synthesized from 500 ng of total RNA with a PrimeScript RT reagent kit and gDNA Eraser (Takara, Tokyo, Japan). Gene expression was determined by quantitative RT-PCR using the SYBR green-based fluorescence method (SYBR Premix Ex Taq, Takara). Amplification was performed using StepOnePlus (Applied Biosystems, Foster City, CA, USA). The PCR cycling conditions were 30 sec at 95°C followed by 40 cycles of 5 sec at 95°C and 30 sec at 58°C/60°C. The results were calculated as the expression of the target gene relative to the expression of the β-actin gene. The sequences of primers used in this study are shown in Table 1.