The DNA template for ROR synthesis was amplified by PCR, which contained an RNA polymerase T7 promoter site upstream of the sequence. DNA was isolated and purified using anion exchange columns (Qiagen) and sequenced to confirm that additional mutations had not been incorporated. In vitro RNA transcription synthesis used the mMESSAGE mMACHINE Kit according to the manufacturer’s instructions (Ambion, USA). RNA was purified by the MEGAclear Transcription Clean-Up Kit (Ambion).
The region encoding G9A was amplified by PCR from pBABE-FLAG-hG9a (Addgene, USA) and cloned into pET28a. Proteins were purified with nickel agarose (Thermo Fisher) and measured by the BCA method (BioRad, USA). The N-terminal FLAG-tag protein was expressed in 293 T and then purified with M2 flag beads (Sigma-Aldrich) and eluted with flag peptide (Sigma-Aldrich). Purified, concentrated proteins were stored at -20 °C in a buffer of 20 mM HEPES, pH 7.0, 100 mM NaCl, 0.5 mM EDTA, and 5 % glycerol.
The region encoding G9A was amplified by PCR from pBABE-FLAG-hG9a (Addgene, USA) and cloned into pET28a. Proteins were purified with nickel agarose (Thermo Fisher) and measured by the BCA method (BioRad, USA). The N-terminal FLAG-tag protein was expressed in 293 T and then purified with M2 flag beads (Sigma-Aldrich) and eluted with flag peptide (Sigma-Aldrich). Purified, concentrated proteins were stored at -20 °C in a buffer of 20 mM HEPES, pH 7.0, 100 mM NaCl, 0.5 mM EDTA, and 5 % glycerol.