4d amaxa nucleofector unit for ebv b cells

We cloned approximately 350 bp of the DNA sequences surrounding the non-risk or risk alleles of selected UBE2L3 or YDJC variants (Supplemental Table 2) into the promoter-less firefly luciferase plasmid, pGL4.14, or the minimal promoter firefly luciferase plasmid, pGL4.23 (Promega). Site-directed mutagenesis was used to separate the physically close risk variants, rs140491 and rs11089620 or rs12484550, rs5998599 and rs9621715 (Supplemental Table 2). The empty vector, non-risk clone, or risk clone was transiently co-transfected with the transfection control renilla luciferase plasmid, pRL-TK, into homozygous non-risk EBV B cells using 4D Amaxa Nucleofector Unit for EBV B cells (Lonza; Nucleofector SF kit, #V4XC-2032). Twenty-four hours post transfection, cells were treated with or without P/I for 2 h. Promoter or enhancer activity was determined according to the Promega Dual-Luciferase Reporter Assay manufacturer instructions (Promega). Relative luciferase units (RLU) for each sample were determined by normalizing the firefly luciferase activity to the renilla luciferase activity. RLU was further normalized to the vector only control and reported as normalized RLU.

Jurkat and THP-1 cells were procured from ATCC. Epstein Barr Virus (EBV)-transformed B cell lines were obtained from the Lupus Family Registry and Repository (LFRR) housed by the Oklahoma Rheumatic Disease Research Cores Center (ORDRCC) at the Oklahoma Medical Research Foundation (OMRF) with IRB approval (22 (link)). Sanger sequencing was used to verify the genotype of EBV B cell lines carrying the risk (A) or non-risk (C) allele of the index SNP, rs140490. Jurkat, THP-1, and EBV B cells were maintained in RPMI 1640 medium supplemented with 10% FBS, 1X penicillin-streptomycin antibiotic mixture (Atlanta Biologicals, Inc.), and 2 mM L-glutamine (Lonza). THP-1 cell medium was also supplemented with 50 μM ß-mercaptoethanol. Where indicated, cells were stimulated with phorbol 12-myristate 13-acetate and ionomycin (P/I; 50 ng/mL, 500 ng/mL) for 2 h prior to harvest. For the siRNA knockdown of CTCF or YY1, EBV B cells homozygous for the UBE2L3-YDJC non-risk or risk haplotype were transiently transfected with 10 nM siRNA using 4D Amaxa Nucleofector Unit for EBV B cells (Lonza; Nucleofector SF kit, #V4XC-2032). The ON-TARGETplus human CTCF siRNA SMARTPool (#L-020165-00-0005), human YY1 siRNA SMARTPool (#L-011796-00-0005), and non-targeting scramble siRNA pool (#D-001810-10-05) were purchased from Dharmacon. All stock laboratory chemicals were from Sigma Aldrich or ThermoFischer.