PMID:31036827 CUT&Tag: https://dx.doi.org/10.17504/protocols.io.x54v9mkmzg3e/v5; CUT&Run: https://dx.doi.org/10.17504/protocols.io.zcpf2vn; OTHER CUT&Run.ChIP: For each experiment, 2.5 × 106 K562 cells were bound to 50 µl of ConA-coated magnetic beads.ncubation with the Pol II-Ser5p primary antibody (Cell Signaling Technology, 13523S), washes and pA–MN binding were performed as described above for CUT&RUN, using five times the corresponding buffer volumes in 1.5-ml low-binding flip-cap tubes. MNase digestion was initiated by resuspending the ConA beads in 500 µl of dig-wash buffer containing 2 mM CaCl₂ and incubating for 2 h at 4 °C. The reaction was stopped by directly adding 500 µl of stop buffer supplemented with Pol II CTD repeat YSPTSPS phospho-Ser5 peptide (Abcam, ab18488) to a final concentration of 20 µg/ml. Samples were incubated for 20 min at 37 °C and centrifuged for 5 min at 16,000g and 4 °C. The 1 ml of supernatant containing released Pol II-Ser5p-bound native chromatin fragments was collected on a magnetic stand and divided into five 200-µl aliquots. One aliquot was retained at 4 °C as the Pol II-Ser5p CUT&RUN input, and the remaining aliquots were used for ChIP. Rabbit monoclonal IgG isotype-control (Abcam, ab172730), anti-NPAT (Invitrogen, PA565419) or anti-RPB3 (Bethyl Laboratories, A303-771A) antibody was added to the corresponding ChIP aliquot at a final dilution of 1:100, and samples were incubated overnight at 4 °C. Protein A Dynabeads (Invitrogen, 10002D) were equilibrated in Tween wash buffer consisting of wash buffer supplemented with 0.05% Tween-20 (Sigma, P7949). 20 µl of Dynabeads was added to each ChIP sample, followed by incubation for 30 min at 4 °C. Beads were washed once with 500 µl of Tween wash buffer and resuspended in 5 µl of release solution containing 10 mM TAPS, 0.1% SDS and Thermolabile Proteinase K at a 1:10 dilution (New England Biolabs, P8111S). Samples were incubated in a thermocycler with a heated lid for 1 h at 37 °C and then for 1 h at 58 °C. SDS was quenched by adding 4 µl of 1.5% Triton X-100 and and 24 μl of ddH2O. DNA from the input and ChIP samples was purified using the DNA Clean & Concentrator-5 Kit (Zymo Research, D4014), eluted in 10 µl of elution buffer according to the manufacturer's instructions and used for library preparation.