Nucleospin plasmid easypure mini kit

Cellular DNA was extracted using the Puregene Kit (Qiagen) according to the manufacturer’s instructions. Five hundred nanograms of DNA were bisulfite converted using the EZ DNA Methylation-Gold Kit (Zymo Research) according to the manufacturer’s instructions. Bisulfite-converted DNA was subjected to methylation-specific PCR using specific primers for the BCL2 promoter listed in Supplementary Table 2. PCR product was resolved on a 2% agarose gel and purified using the Monarch DNA Gel Extraction Kit (New England BioLabs), cloned into pCR4-TOPO TA Vector (Thermo Fisher Scientific), transformed into XL10-Gold Ultracompetent Cells (Agilent Technologies), and plated onto ampicillin selection LB-agar plates. DNA of single colonies was extracted using the NucleoSpin Plasmid EasyPure Mini Kit (Macherey-Nagel) and submitted to Sanger sequencing using sequencing primers M13-for and M13-rev (see Supplementary Table 2). Obtained sequences were analyzed and DNA methylation plots were generated using the QUMA quantification tool for methylation analysis^{43 (link)}.

The pCMV3 expression vector encoding human wild-type ADNP fused to either an N-terminal GFPSpark® or N-DYKDDDDK (Flag®) tag was purchased from Sino Biological (HG11106-ANG; HG11106-NF). The c.1676duplA mutation was introduced in the N-DYKDDDDK (Flag®) ADNP expression vector by PCR mutagenesis using the Q5® Site-Directed mutagenesis kit (New England Biolabs; E0554S) according to manufacturer’s protocol. Mutagenesis primers were designed using the NEBaseChanger Tool (http://nebasechanger.neb.com/). The mutation was inserted with the forward primer: 5′-ACACTAACATCCATCTCCTG-3’ and the reverse primer: 5′-TGACTACCCTGCTGCAAT-3′ by thermocycling with an annealing temperature of 60 °C. DNA was purified from transformed high-efficiency NEB 5-alpha competent E. coli cells using the NucleoSpin Plasmid EasyPure Mini kit (Macherey Nagel; 740727.50) according to the manual. The mutation was confirmed by Sanger sequencing.