Pcpgfree lucia

Luciferase reporter assays were carried out as described previously [42 (link)]. Briefly, genomic regions of interest were amplified by PCR from dermal fibroblast genomic DNA and cloned into pGL4.10, pGL4.23 (Promega), or pCpGfree-promoter-lucia (Invivogen). Open reading frames were obtained from the Genomics and Proteomics core facility (DKZF) and cloned into pDest11-based Gateway expression vectors (Thermo Fisher Scientific). Reporter constructs and open reading frames were validated by Sanger sequencing (GATC Biotech, Constance, Germany). All cell lines were transfected with 40 ng of reporter plasmid and 5 ng of open reading frame plasmid using TransIT-LT1 transfection reagent (Mirus Bio) in 384-well plates. Readout was carried out 48 h after transfection. Data were normalized to co-transfected luciferase reporter vectors (pRL-TK-renilla luciferase (Promega) for pGL4-based reporters and pGl4-CMV-firefly luciferase for pCpGfree-lucia reporters). In vitro methylation of reporters was carried out using M.SssI CpG methyltransferase (Thermo Fisher Scientific).

Genomic regions-of-interest were amplified by PCR from dermal fibroblast DNA and cloned into pGL4.10, pGL4.23 (both Promega) or pCpGfree-promoter-lucia (Invivogen). Reporter constructs were validated by Sanger sequencing (GATC Biotech). Site-directed mutagenesis was carried out according to standard protocols. HEK293T cells were transfected with TransIT-LT1 transfection reagent (Mirus Bio) and readout was carried out 48 h after transfection. Data were normalized to co-transfected luciferase reporter vectors (pRL-TK-renilla luciferase (Promega) for pGL4-based reporters and pGl4-firefly luciferase for pCpGfree-lucia reporters). In vitro methylation of reporters was carried out using M.SssI CpG methyltransferase (Thermo Scientific).