Dynabeabs myone streptavidine c1

The iPOND experiment was performed as described elsewhere51 with minor modifications. Briefly, 10⁸ cells were pulse-labelled with 10 μM EdU (Invitrogen) for 10 min. Immediately after the pulse or after a 1 h chase in fresh medium supplemented with 10 μM thymidine (Sigma), cells were crosslinked with 1% formaldehyde (Sigma) in PBS for 15 min at room temperature under gentle agitation. Crosslink was stopped by addition of 125 mM glycine for 5 min. Cells were harvested by scrapping and washed in ice-cold PBS. Pellets were permeabilized in PBS with 0.5% Triton × 100 for 30 min at room temperature. Biotin-azide (Molecular Probes) was conjugated to EdU by click chemistry for 2 h in click reaction buffer (10 mM sodium-L-ascorbate, 10 μM Biotin-azide, 2 mM CuSO₄ in PBS). Cells were lyzed in iPOND lysis buffer (10 mM Hepes-NaOH pH 7.9, 100 mM NaCl, 2 mM EDTA, 1 mM EGTA, 1 mM PMSF, 0.2% SDS, 0.1% sarkozyl, antiproteases) and sonicated on a Bioruptor device (30 cycles of 30 s on/30 s off at the highest setting). Solubilized chromatin was retrieved by centrifugation at 16,000g for 10 min and supernatant was further incubated overnight with magnetic streptavidin beads (Dynabeabs MyOne Streptavidine C1, Invitrogen). Beads were washed once in lysis buffer, once in 500 mM NaCl and twice in lysis buffer. Proteins were eluted by boiling in 1 × Laemmli buffer at 95 °C for 30 min.