Dharmafect 1

Human pancreatic islets were dissociated using Cell Dissociation Buffer enzyme-free, Hanks’ Balanced Salt Solution (ThermoFisher Scientific. Burlington, ON). Dissociated human islets cells and INS 832/13 cells were transfected with scramble siRNA (SiScr) and siRNA against secretagogin (SiScgn) using DharmaFECT 1 (GE Healthcare) according to manufacturer’s protocol (see Supplemental Materials for additional details). In patch-clamp studies, a fluorescent siRNA (Cat # 1027284, Qiagen, Toronto, Canada) was included to identify transfected cells. INS 832/13 cells or dispersed human islet cells were transfected with GFP or GFP-tomosyn1 plasmids (generously given by Dr. Romano Regazzi, University of Lausanne, Switzerland) using Lipofectamine 2000.

The UT-SCC 16A and 16B and UTSCC 74A and 74B cells were seeded in media without antibiotics (1.2 × 10⁵cells/well) and treated with siRNA SETDB1 using a transfection reagent (DharmaFECT-1, GE Healthcare, USA). The efficiency of the transient transfection in cells treated with siRNA SETDB1 was assessed by qRT-PCR and western blotting. The manufacturer’s protocol was followed. After 24 h, the cells were harvested for further analyses.
For transient transfection by siRNA knockdown, siRNApool technology was used, and all of the siRNAs were synthesized by Dharmacon (GE Healthcare, USA). For specific siRNAs SETDB1, for a nonsilencing control and GAPDH control, the ON-TARGETplus Human SETDB1 siRNA-SMARTpool and Human Non-Targeting-Control Pool and Human GAPDH-Control Pool (GE Healthcare, USA) were respectively used (Özdaş, 2018).

HT-29 cells in 24-well plates were transfected with siRNA (2.5 μl of 5 μM) using DharmaFECT1 (1 μl per reaction) reagent (GE Life Sciences). All siRNAs used in this study were SMARTpool ON-TARGET siRNA purchased from Dharmacon (Supplementary Table 1). Canonical cGAMP (2’3’ cGAMP) (8 μg/ml) was transfected into HT-29 and HEK293 cells using lipofectamine 2000 as previously described^{42 (link)}. 2’2’ cGAMP (Invivogen) was used as a negative control for which no IFN induction was observed.

DNA was transiently transfected into cells using FuGENE 6 transfection reagent (Promega), according to the manufacturer's instructions. The gRNAs for CRISPR genome editing were cloned into the CRISPR-Cas9 plasmid px330. The gRNAs used in this study were VPS35, 5′-GTGGTGTGCAACATCCCTTG-3′ and SNX27, 5′-GGCTACGGCTTCAACGTGCG-3′. CRISPR-Cas9 plasmids were co-transfected with a puromycin resistance-expressing plasmid, and cells were subjected to puromycin selection 24 h later.
For siRNA-based knockdown, cells were reverse-transfected using DharmaFECT 1 (GE Healthcare) and then transfected again 24 h later according to the manufacturer's instructions. At 48 h after the second transfection, cells were lysed or fixed and processed for immunofluorescence. For VPS35 suppression, a combination of oligonucleotides 3 (sequence 5′-GUUGUUAUGUGCUUAGUA-3′) and 4 (sequence 5′-AAAUACCACUUGACACUUA-3′) of the Dharmacon ON-TARGETplus VPS35 siRNA SMARTpool was used. For SNX27 suppression, Dharmacon ON-TARGETplus SNX27 siRNA SMARTpool (cat. #L-017346-01) was used. The non-target control siRNA used was Dharmacon ON-TARGETplus non-targeting siRNA no. 2 (cat. #D-001810-02).