Quantification of XBP1 Splicing

RNA was extracted and reverse transcribed as previously described (Cox et al., 2011 ). Protocols for quantification of splicing of XBP1 were described previously (Cox et al., 2011 ; Brown et al., 2016 (link)). qPCRs were run on a Rotorgene 3000 (Qiagen, Crawley, UK) using GoTaq G2 Flexi DNA polymerase (Promega, Southampton, UK; cat. no. M7801) and analyzed as described before (Brown et al., 2016 (link)). Primer sequences are listed in Table 2 or have been reported before (Brown et al., 2016 (link)). Amplification of a single PCR product was confirmed by recording the melting curves after each PCR. The amplification efficiencies for all qPCRs were ∼0.75 ± 0.05. Results represent the average and SE of three technical repeats. qPCR results were confirmed by at least one other biological replicate.