Retroviruses for Epigenetic Enzyme Knockdown

To construct retrovirus-based short hairpin RNA (shRNA) expression vectors for knockdown of epigenetic demethylases, two oligonucleotides containing shRNA sequences (Supplementary Table S1) were annealed and inserted into a mouse U6 promoter-driven vector pMXs-U6 puro (provided by Dr Toshio Kitamura) (26 (link)). Expression plasmids of individual enzymes (Supplementary Table S2) were constructed using a long terminal repeat promoter-driven expression vector pMXs-puro (from Dr. T. Kitamura), in which the original puromycin-resistant gene was replaced with the blasticidin S-resistant gene. The iron-binding site of each enzyme sequence was modified by a previously reported mutation by the PCR-based site-directed mutagenesis (27–43 (link)) (Supplementary Table S2). Using the similar method, shRNA-resistant plasmids were prepared by introducing silent mutations in the targeted sequence (Supplementary Table S3). Retroviruses were produced by transfecting each plasmid into Platinum-E packaging cells (from Dr T. Kitamura) using polyethyleneimine MAX (Polyscience, 24765-1). Retrovirus infection was performed as reported previously (25 (link)) and the infected cells were selected by 10 μg/ml puromycin (Thermo Fisher, ant-pr-1) or 10 μg/ml blasticidin S (Thermo Fisher, ant-bl-1).