Basic pnl vector

The selected promoters and enhancer sequences analysed are listed in Table 1. Each fragment was PCR amplified and cloned into the basic pNL-vector (Promega) upstream of the NanoLuc luciferase reporter gene. For promoter assays, the fragments were cloned into KpnI/HindIII sites of the promoterless pNL plasmid. For enhancer assays, the fragments were first inserted upstream of the minimal promoter element into the KpnI/HindIII sites of the pNL-minP NanoLuc luciferase reporter plasmid (Promega). Subsequently, the fragment (Xist bp 2,000–1,380) that displayed the highest enhancer activity was cloned upstream of the putative XistAR promoter sequence (Xist bp 3,000–2,750). The plasmids were transfected into female XEN cells plated in a 96-well format. Each well was transfected with 100 ng of pNL-NanoLuc reporter construct and 6 ng of transfection control plasmid encoding the firefly luciferase gene (pGL3-firefly; Promega), with Lipofectamine 2000 (Invitrogen) transfection reagent, following the manufacturer's instructions. Each construct was tested in triplicate in each of three separate transfections. Seventy-two hours after transfection, the activities of firefly and NanoLuc luciferase were measured using the Nano-Glo Dual-Luciferase reporter assay system (Promega, #N1610). The NanoLuc luciferase activity was normalized with firefly luciferase activity for each transfection.