Wheat Germ Extract Chromatin Assembly

Chromatin assembly grade wheat germ extract (WEPRO7240Ch, CellFree Sciences) was used to co-express all core histone combinations using approximately 2 μg each histone mRNA^{31 (link)}. As a chromatin assembly control, 2 μg each of canonical H3 or variant H3 and H4-coding mRNA was used for the reaction. Briefly, individually transcribed and purified mRNAs were used to make 5 μL premix mRNA solutions for each chromatin combination and were mixed with 5 μL wheat germ extract, 0.4 μL of 1 mg·mL⁻¹ creatine kinase, 0.5 μL of 0.5 μg·μL⁻¹ pBSK plasmid, 0.1 μL of 20 U·μL⁻¹ Topoisomerase I (Takara, Shiga, Japan), and adjusted with nuclease-free water to 10.7 μL. Topoisomerase I was added in order to release torsional strain during chromatin assembly onto circular closed plasmid. The reaction mixture was carefully layered under 103 μL of 1xSUB-AMIX (CellFree Sciences) to create a bilayer in a sterile microplate well. The chromatin assembly reaction was performed for 4 h at 26 °C.