Quantitative PCR Verification of UPR Transducers

Quantitative (q)RT-PCR was performed on transcripts of interest that were identified in the RNA-Seq dataset using procedures we have been previously described [24 (link)]. Gene-specific primers are shown in Supplementary Table 1. We verified 9 mRNAs associated with the three primary transducers of the UPR (Supplementary Figure 1). Total RNA was isolated using the RNEasy fibrous tissue kit according to the manufacturer’s instructions. RNA quantity and purity were assessed by spectrophotometric analysis (Nanodrop) in which both the ratios of absorbance at 260 nm to that at 230 nm (A260/230) and the absorbance at 260 nm to that at 280 nm (A260/280) were >1.8. cDNA synthesis was performed using SuperScript III First-Strand Synthesis System for RT-PCR cDNA Synthesis Kit (Invitrogen, according to the manufacturer’s protocol. The cDNA-equivalent of 5 ng RNA was used for amplification in 384-well microtiter plates in a QuantStudio 7 cycler (Applied Biosystems, CA, USA) using SYBR green assays. Cycle threshold (CT) values for individual reactions were normalized against β2 microglobulin expression. All cDNA samples were amplified in duplicate. Relative expression was calculated using the ∆CT method. Data are presented as fold change compared with control, obtained using the ∆∆CT method.