Gotaq g2 hot start taq polymerase

624-38 XBP1KO and B16F10-OVA XBB1KO were obtained using XbpI human and mouse genes knockout kits respectively according to manufacturer’s instructions (Cat# KN401959 and #KN519483, Origene, Rockville, MD, USA). Stable KO cells were selected in complete medium containing 2 μg/ml puromycin for 2 week. Single cell clones were isolated and screened by touchdown PCR. Primers were designed as indicated in Figure S5 and are listed in Table S3. Touchdown PCR were performed using GoTaq G2 Hot Start Taq Polymerase (Promega) and the following cycling conditions: 2 min at 95°C, followed by a touchdown phase of 10 cycles at 95°C for 30 s, 65-55°C (dropping 1°C per cycle) for 30 s and 72°C for 1 min; 25 cycles at 95°C for 30 s, 55°C for 30 s and 72°C for 1 min; 72°C for 5 min. For B16F10-OVA XbpIKO clones, two successive runs of PCR have been performed to obtain enough product to be sequenced. Then PCR products were purified and sequenced. Knockout of protein in both cell lines was confirmed by western blot: Rabbit mAb anti XBP1 s (Cell Signaling, 1:1000).

XBP1 splicing in MyPs was measured by semiquantitative PCR analysis. RNA was extracted and cDNA synthesized as described for RNA sequencing. Semiquantitative PCR was performed with 1 ng cDNA using GoTaq G2 Hot Start Taq Polymerase (Promega) under the following conditions: initial denaturation at 94°C for 90 s, followed by 38 cycles of 94°C, 30 s; 64°C, 45 s; and 72°C, 45 s. Primer sequences were as follows: 5′-TTA​CGG​GAG​AAA​ACT​CAC​GGC-3′ and 5′-GGG​TCC​AAC​TTG​TCC​AGA​ATG​C-3′. PCR products were separated on a 4% agarose gel, and band intensity was measured with ImageJ.