Random hexamers

Total RNA was extracted from tumor tissue by using Trizol Reagent (Thermo Fisher Scientific) according to the manufacturer's protocol. RNA quality was confirmed by electrophoresis on a 1% agarose gel in 2× RNA loading dye (Thermo Fisher Scientific). For genomic DNA removal, the RapidOut DNA Removal Kit (Thermo Fisher Scientific) was used. For cDNA synthesis, the SuperScript II Reverse Transcription Kit (Thermo Fisher Scientific) with random hexamers (Microsynth, Balgach, Switzerland) was employed. Real‐time quantitative polymerase chain reaction (RT‐qPCR) analysis was performed on a Bio‐Rad CFX96 (Hercules, CA) using the Brilliant III Ultra‐Fast Quantitative PCR Kit (Agilent technologies, Santa Clara, CA) and TaqMan probes. Probes and primers were purchased from Thermo Fisher Scientific (CCL2 Mm00441242_m1, CCL3 Mm00441259_g1, CCL4 Mm00443111_m1, CXCL9 Mm00434946_m1, CXCL10 Mm00445235_m1, Galectin‐9 (Lgals9) Mm00495295_m1, Gp100 Mm00498996_m1, H60b Mm04243254_m1, H60c Mm04243526_m1, IL‐10 Mm00439614_m1, IL‐12a Mm00434165_m1, IL‐12b Mm01288989_m1, IL‐15 Mm00434210_m1, IL‐18 Mm00434225_m1, PD‐L1 Mm00452054_m1, PD‐L2 Mm00451734_m1, Rae1 Mm00558293_g1, TGF‐β1 Mm01178820_m1, Trp‐2 Mm01225584_m1, Ulbp1 Mm01180648_m1). Sequences for probes and primers specific for TATA binding protein (Tbp) were synthesized by Microsynth.

RNA was transcribed into cDNA using random hexamers (Microsynth, Balgach, Switzerland) and SuperScript III reverse transcriptase (cDNA synthesis kit; Invitrogen) according to the manufacturer's protocol. Isopropanol precipitation was performed to prepare DNA and cDNA samples for storage and shipment (73 ).

HEK293T cells were seeded overnight and RNA was transfected using Lipofectamine 2000 according to manufacturer’s instructions. Cells were lysed at the indicated time point using TRIzol™ Reagent (15596026, Thermo Fisher Scientific), RNA extraction was performed according to the manufacturer’s instructions. RNA was reverse transcribed using the TaqMan™ MicroRNA Reverse Transcription Kit (4366597, Applied Biosystems™). The reverse transcription reaction with the end concentration of: 1× RT buffer, 1× dNTP mix (4 mM), 12.5 μM random hexamers (Microsynth), 12.5 μM oligo(dT)15 (C1101, Promega), 2.5 U multiscribe Reverse transcriptase, 2 U RNAse inhibitor (RNasin, N2115, Promega) was run on C1000 or S1000 Thermal Cycler (Bio-Rad) using the following cycle: 25°C for 10 min, 37°C for 120 min, 85°C for 5 min, 10°C on-hold. Transcript specific primers (Supplementary Table S3) were ordered from Microsynth (Balgach, Switzerland). The SYBR Green PCR was performed in a LightCycler 480 instrument (Roche) with KAPA SYBR® FAST for Roche LightCyler®480 (KK4610, Sigma-Aldrich) following the manufacturer’s instructions. Fold changes are calculated using the 2^−ΔΔCp method. Housekeeping genes were used for normalization and mock/negative control treated cells as calibrators.