DNA fragments were synthesized from IDT (Integrated DNA Technologies)
and loaded into the pGL3-Promoter Vector (Promega, E1761).
1×106 Jurkat cells were resuspended in 100μl of
nucleofector solution mixture (Lonza, Amaxa cell line nucleofector kit V), then
1.5μg of each reporter vector and 0.5μg of pRL-TK Renilla control
luciferase reporter vector (Promega) were added. Cells with reporter plasmid DNA
were electroporated into Jurkat cells using program X-005 on Lonza Nucleofector
2b (Lonza) and resuspended in 2ml of RPMI medium supplemented with 10% FCS and
penicillin-streptomycin. After being incubated at 37°C/5%CO2 for 48
hours, cells were collected by centrifugation and luciferase activities were
measured using the Dual-Luciferase Reporter Assay system (Promega, E1960). For
each putative enhancer, experiments were performed in triplicates and replicated
independently for 3 or 2 times (Supplementary Note ). Cell numbers
and transfection efficiency were normalized to Renilla luciferase activity.
and loaded into the pGL3-Promoter Vector (Promega, E1761).
1×106 Jurkat cells were resuspended in 100μl of
nucleofector solution mixture (Lonza, Amaxa cell line nucleofector kit V), then
1.5μg of each reporter vector and 0.5μg of pRL-TK Renilla control
luciferase reporter vector (Promega) were added. Cells with reporter plasmid DNA
were electroporated into Jurkat cells using program X-005 on Lonza Nucleofector
2b (Lonza) and resuspended in 2ml of RPMI medium supplemented with 10% FCS and
penicillin-streptomycin. After being incubated at 37°C/5%CO2 for 48
hours, cells were collected by centrifugation and luciferase activities were
measured using the Dual-Luciferase Reporter Assay system (Promega, E1960). For
each putative enhancer, experiments were performed in triplicates and replicated
independently for 3 or 2 times (
and transfection efficiency were normalized to Renilla luciferase activity.