T4 pnk m0201s

tet-pLKO-shEZH2 was constructed by inserting the EZH2 shRNA (the sense sequence is 5′-TATTGCCTTCTCACCAGCTGC -3′) into the tet-pLKO-puro vector (Addgene) digested with AgeI and EcoRI restriction enzymes (NEB) and dephosphorylated for 30 min at 37 °C. The digested plasmid was run on a 1% agarose ge l, cut out, and purified using the Wizard SV Gel and PCR Clean Up kit (Promega). The oligonucleotides were phosphorylated using T4 PNK (M0201S) with T4 Ligation Buffer (New England Biolabs, Inc.). Samples were annealed in a thermocycler at 37 °C for 30 min and then at 95 °C for 5 min and finally were ramped down to 25 °C at 5 °C per min. Annealed oligonucleotides were diluted 1:200 in RNase/DNase-free water. Ligation of the annealed oligonucleotide and digested tet-pLKO-puro plasmid was performed using Quick Ligase (New England Biolabs, Inc.). To induce EZH2 knockdown, cells infected with tet-inducible shEZH2 virus were treated with 100 ng/ml of Doxycycline (Sigma).

pLentiCRISPR-CARM1 was constructed by inserting the CARM1 guide RNA (gRNA; 5′-AGCACGGAAAATCTACGCGG-3′). pLentiCRISPR v2 (Addgene) was digested and dephosphorylated with BsmBI restriction enzyme (Fermentas) for 30 min at 37 °C. The digested plasmid was run on a 1% agarose gel, cut out, and purified using the Wizard SV Gel and PCR Clean Up kit (Promega). The oligonucleotides were phosphorylated using T4 PNK (M0201S) with T4 Ligation Buffer (New England Biolabs, Inc.). Samples were annealed in a thermocycler at 37 °C for 30 min and then at 95 °C for 5 min and then were ramped down to 25 °C at 5 °C/min. Annealed oligonucleotides were diluted 1:20 0 in RNase/DNase-free water. Ligation of the annealed oligonucleotide and digested pLentiCRISPR v2 plasmid was performed using Quick Ligase (New England Biolabs, Inc.).