Nanoluc luciferase reporter plasmid

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.

To generate the JUN 5′ UTR and the HBB 5′ UTR Nluc reporter plasmids, the JUN 5′ UTR (ENST00000371222.4) previously generated by amplification from human cDNA [18 (link)] and the HBB 5′ UTR (ENST00000335295.4) commercially generated (IDT) sequences were each inserted into the pNL1.1 NanoLuc luciferase reporter plasmid (Promega, GenBank Accession Number JQ437370) downstream of a T7 promoter using overlap-extension PCR with Q5 High-Fidelity DNA Polymerase (NEB) and InFusion cloning (Takara Bio). For the JUN AUG mutants, the first 51 nucleotides of the JUN CDS were inserted downstream of the full JUN 5′ UTR sequence and upstream of the full Nluc CDS in the pNL1.1 plasmid. For the Fluc reporter plasmid, the HBB 5′ UTR Nluc reporter plasmid was amplified and the NanoLuc luciferase sequence was replaced by a commercially generated Firefly luciferase sequence (IDT) [71 (link)]. Subsequent mutant versions of the JUN reporter plasmids were made by amplifying the plasmid using overlap-extension PCR with Q5 High-Fidelity DNA Polymerase (NEB) and primers containing the corresponding mutations, insertions, or deletions, followed by InFusion cloning (Takara). All primers used for amplification can be found in S2 Table. All sequences were verified by Sanger sequencing. Protocol available: http://dx.doi.org/10.17504/protocols.io.kqdg3xoyzg25/v1.[PROTOCOL DOI].