Neb4 buffer

Two micrograms of reconstituted nucleosome arrays were incubated with 0.2 micrograms recombinant protein for 10 minutes at room temperature in 1X NEB4 buffer (New England Biolabs). AvaI restriction enzyme (12U per timepoint) was added and the reaction quenched on ice in 80% PCR cleanup binding buffer. Timepoints were subjected to PCR cleanup (Bio Basic, cat#BS664) and visualized via 0.7% agarose gel electrophoresis in 0.2X TB.

1 μl containing 100 ng genomic DNA, extracted as previously annotated, was mixed with 7.1 μl H₂O and 0.9 μl NEB 4 buffer (NEB). After incubating for 10 min at 96°C, the mix was cooled down to 4°C and supplemented with 1 μl of a solution containing 4 U Terminal Transferase (Thermo Scientific), 0.1 μl 10× NEB 4 buffer (NEB), 0.1 μl 10 mM dCTP and H₂O. The samples were then incubated for 30 min at 37°C, 10 min at 65°C and 5 min at 96°C. Afterwards, the samples were kept at a temperature of 65°C. Meanwhile, 21 μl H₂O were mixed with 4 μl 10× PCR buffer (89.11 mM Tris–HCl pH 8.8, 21.28 mM (NH₄)₂SO₄, 6.65% glycerol and 0.0133% Tween-20), 4 μl dNTPs (2 mM stock), 0.3 μl forward subtelomeric oligo (100 μM stock), 0.3 μl G₁₈ reverse oligo oBL359 (100 μM stock) and heated at 65°C. At this temperature, 0.5 μl Phusion Hot Start II DNA Polymerase (Thermo Scientific) were added and the resulting mix was combined with the samples at 65°C. Thermocycling took place as follows: 3 min at 95°C, 45 cycles of 30 s at 95°C, 15 s at 63°C and 20 s at 68°C. At the end, the samples were held for 5 min at 68°C and then at 4°C. The derived PCR products were separated on a 1.8% agarose gel for 2.5 h at 100V. The telomere length was defined by Image Lab or ImageJ.