Cell line kit 5

Reprogramming of LCLs (approximately 1 × 10⁶ cells) with 2 μg Epi5™ Episomal Reprogramming plasmids (Life Technologies Inc., Carlsbad, CA) was performed via electroporation using the Amaxa Nucleofector Device II, Cell Line Kit V, and program T-020, following the Cell Line Kit V protocol (Lonza, Basel, Switzerland). The Epi5™ plasmids contain reprogramming factors Oct3/4, Sox2, Klf4, Lin28, and L-myc and two additional factors, mp53DD and EBNA, to aide in cell cycle stabilization. After transfection, each sample was transferred directly into 1 well of a 6 well plate coated with hESC-qualified Matrigel (1:60; BD Biosciences, San Jose, CA) with LCL media and incubated overnight. Twelve hours post-transfection the Epi5™ Episomal Reprogramming (“Reprogramming CD34⁺ Cells (Feeder-free culture)”) protocol was followed starting at Day 1 and continuing until mature iPSC colonies were apparent (at Day 15–20). Once iPSC colonies were present, it was necessary to perform an Essential 8 media (Life Technologies, Inc.) change every day to prevent differentiation. At passage 5, all clonal lines were transitioned to mTeSR-1 medium (StemCell Technologies, Vancouver, BC).

The siRNA sequences used for silencing STING, Lyn, AIM2, cGAS, LRRFIP1 and ZBP1 were purchased from RIBOBIO (China). The nucleofector device (Amaxa, Germany) and cell line kit V (Amaxa Biosystems, USA) were used to transfect siRNA duplexes into BJAB cells. Negative control of siRNA was transfected as matched control. Cells were harvested at 36th hour after transfection and used for subsequent experiments.

Human STING cDNA was PCR-amplified using the primer pair: FP: 5-CCCAAGCTTGATGCCCCACTCCAGCCT-3, RP: 5-CCGCTCGAGCTCAAGAAATCCGTGC-3. The product was cloned into the BamH1/HindIII sites of pcDNA3.1-Myc-His (Invitrogen, USA), resulting in STING-containing pcDNA3.1. Then STING-containing pcDNA3.1 plasmid (2 μg) was transfected into BJAB cells using the nucleofector device (Amaxa, Germany) and cell line kit V (Amaxa Biosystems, USA). Empty vector were transfected as matched control. At 36th h after transfection, cells were harvested and used for subsequent experiments.