Uv vis spectrophotometer

Total RNA was extracted from PBMCs with TriPure Isolation Reagent (Roche Diagnostics GmbH, Mannheim, Germany) and the concentration of RNA was determined by measuring the absorbance at 260 nm in a UV–Vis spectrophotometer (Quawell, San Jose, CA, USA). Reverse transcription was performed by the Bio-Rad Systems (Bio-Rad, Hercules, CA, USA) according to standard protocols using the Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics GmbH). The expression level of Gαq was measured by real-time quantitative PCR. β-actin was simultaneously amplified and used as an internal control. The primer sequences were as follows: β-actin forward, 5′-AGAAAATCTGGCACCACACC-3′; β-actin reverse, 5′-AGAGGCGTACAGGGATAGCA-3′; Gαq forward, 5′-GTTGATGTGGAGAAGGTGTCTG-3′; and Gαq reverse, 5′-GTAGGCAGGTAGGCAGGGT-3′. Amplification was performed with the 7500 Real Time PCR Systems (Applied Biosystems, CA, USA). Gene expression levels were normalized by comparing to β-actin and relative expression was calculated by the2^–ΔΔCt method.

Total RNA of each sample was isolated separately using Trizol Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. The purified RNA concentration was quantified by a spectrophotometer (UV-Vis Spectrophotometer, Quawell Q5000, San Jose, CA, USA), and the purity and degradation of total RNA were checked on 1% agarose gels before proceeding. For maximizing the diversity of transcriptional units, RNA from each sample was mixed into a single pool. The mRNA-seq library was constructed using Illumina’s TruSeq RNA Sample Preparation Kit (Illumina Inc, San Diego, CA, USA). Shortly, mRNA was purified by oligo (dT) magnetic beads. After purification, the mRNA was fragmented into small pieces using divalent cations under elevated temperature and the cleaved RNA fragments were used for first strand cDNA synthesis using reverse transcriptase and random primers. This was followed by second strand cDNA synthesis using DNA polymerase I and RNaseH. Then, cDNA fragments were perfored an end repair process and ligation of adapters. The product was purified and enriched with PCR to create the final cDNA library [26 (link)]. Finally, the cDNA library was sequenced by the Illumina HiSeq^™ 2000 sequencing platform.