Pgl4.23 firefly luciferase vector

Human neuroblastoma BE(2)-C cells were plated at 2×10⁴ cells/well in 96-well plates and 24 hours later were transfected with Lipofectamine 3000 (L3000–015, Thermofisher Scientific) together with 75ng of Promega pGL4.23 firefly luciferase vector containing the 230nt of human genomic DNA from the loci of interest (Supplementary Table 6), and 4ng of pNL3.1 NanoLuc (shrimp luciferase) plasmid, for normalization of transfection conditions. 42 hours after transfection, luminescence was detected with the Promega NanoGlo Dual Luciferase assay system (N1630) and BioTek Synergy plate reader. Four to six wells per variant were tested in each experiment. Variants were tested in at least two separate experiments. For each sequence tested, the ratio of firefly luminescence (ASD allele) to NanoLuc luminescence (transfection control) was calculated and then normalized to empty vector (pGL4.23 with no insert) on the same plate. Statistics were calculated from fold over empty vector values from each experiment and results from multiple replication experiments are combined with Fisher’s combined probability test. For presentation of the data, we normalized the fold over empty vector value of the proband allele to that of the sibling allele.

The DNA fragments of E953 and E245 enhancers of Sp9 gene were created by PCR and subsequent cloned into pGL4.23 firefly luciferase vector (Promega) upstream (U) or downstream (D) of Luc2 gene (e.g. pGL4.23-E953U or pGL4.23-E953D). The putative Sp9 promoter was amplified by PCR and cloned into pGL4.10 promoterless firefly luciferase vector (Promega). Mouse embryonal carcinoma cell line P19 were grown in medium MEMα (Gibco 12571-063) supplemented with 10% fetal bovine serum (FBS, Gibco 10099-141). For luciferase assay, P19 cells transfections were performed in triplicate in 24-well plates by using Fugene HD transfection regent according to the manufacturer's protocol (Promega, E2311). Luciferase sparks were quantified by microplate luminometer (Turner BioSystems, Inc. ModulusTM Microplate Reader).