Chromatin Fragment Adapter Ligation Protocol

The bead-nuclei mixture was again pelleted at 2500g for 60 s, and resuspended in 30 μL H₂O, 20 μL biotinylated bridge-adaptor (see Ma et al [23 (link)] for sequences and adaptor preparation), 20 μL blunt bridge-adaptor, 10 μL 10× T4 DNA Ligase Buffer with ATP, 10 μL polyethylene glycol (PEG)-4000 (Thermo), 5 μL 10 % Triton-X100, and 5 μL T4 DNA Ligase (5 U/μL; Thermo). This mixture was incubated at 16 °C overnight to ligate T-tailed biotinylated bridge adapters to the termini of A-tailed, digested chromatin. Following incubation, the reaction was stopped by adding 5 μL 10 % SDS. The bead-nuclei mixture was then pelleted at 2500g for 60 s and resuspended in 300 μL H₂O. To remove excess unligated adapter, 250 μL 20 % PEG in 2.5 M NaCl was added to the mixture, which was incubated at room temperature for 5 min, collected via DynaMag, and washed once with 80 % ethanol. Beads were then resuspended in 200 μL H₂O and purified further using 0.8 volumes of 20 % PEG in 2.5 M NaCl as above, to further remove unligated adaptors.