Cfx connect thermal cycler system

Total RNA from primary WT VSMC, WT MΦ, and WT DC treated as described above was isolated using GeneMATRIX Universal RNA Purification Kit (EURx, E3598). RNA-seq libraries were prepared from at least three biological replicates using a TruSeq RNA Library Preparation kit (Illumina, RS-122) according to the manufacturer's protocol. Libraries were quantified by Qubit fluorometer (TFS) and the quality was assessed with Agilent High Sensitivity DNA kit (Agilent Technologies, 5067-4626). Libraries were sequenced with Illumina HiScanSQ sequencer. To validate the quality of RNA-seq dataset, primary WT VSMC, WT MΦ, and WT DC were treated as described previously and 1 μg of RNA was used to synthetize complementary DNA with RevertAid Reverse Transcriptase (TFS, EP0441). Cxcl9, Cxcl10, Ccl5, Nos2, Gbp6 transcripts were quantified using Maxima SYBR Green/ROX qPCR Master Mix (TFS, K0223) and CFX Connect Thermal Cycler System (Bio-Rad). Target gene levels were normalized to β-actin (ACTB) and quantified as described elsewhere (28 (link)) (described in Results section; data not shown).

Total RNA was isolated using TRI-REAGENT (TRI118, MRC) followed by a column-based Total RNA Zol-Out™ D kit (043, A&A Biotechnology) following the manufacturer’s protocol.
500 ng of total RNA was subjected to reverse transcription according to the protocol of the RevertAid First-Strand Synthesis Kit (K1622, TFS). Transcript quantification was performed by qPCR with Maxima SYBR Green/ROX qPCR Master Mix (K0223, TFS) on the CFX Connect Thermal Cycler System (Bio-Rad). Target gene levels were normalized to the housekeeping gene—glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data were transformed as described previously [21 (link)]. Forward and reverse primers used in experiments are depicted in Table S1. If not indicated differently, results are presented as mean ± SEM for two independent biological repeats. Graphs are prepared using GraphPad Prism 8.3.0.