The selected TREs were individually cloned into eSTARR-seq assay vectors via LR reactions and the resulting library of plasmids was extracted with the E.Z.N.A. Endo Free Plasmid Mini Kit II (Omega Bio-tek, D6950). The plasmids were electroporated into K562 cells with Ingenio Electroporation Kit (Mirus, MIR 50115). For each electroporation, 0.5 million cells were mixed with 1-2 μg plasmids and 50 μl Ingenio Electroporation Solution and electroporated with a Nucleofector II device using Program T-016. The pGL4.75 vector (Promega, E6931) was co-electroporated (10 ng/electroporation) as the internal control. The electroporated K562 cells were recovered in 2 ml culture medium at 37°C with 5% CO2 until harvest.
The electroporated cells were harvested after 24 hours of recovery for dual luciferase assay. The assay was carried out with Dual-Glo Luciferase Assay System (Promega, E2920) according to the manufacturer’s instruction. An Infinite M1000 Microplate Reader (Tecan, 30034301) was used to quantify the luminescent signals. Cells electroporated with only pGL4.75 vector or with only pDEST-hSTARR-luc-Pmyc vector were used as the background controls for firefly or Renilla luciferase activities, respectively.
The electroporated cells were harvested after 24 hours of recovery for dual luciferase assay. The assay was carried out with Dual-Glo Luciferase Assay System (Promega, E2920) according to the manufacturer’s instruction. An Infinite M1000 Microplate Reader (Tecan, 30034301) was used to quantify the luminescent signals. Cells electroporated with only pGL4.75 vector or with only pDEST-hSTARR-luc-Pmyc vector were used as the background controls for firefly or Renilla luciferase activities, respectively.