GSM7032282: H3K36me3-KO-1; Mus musculus; OTHER
Cut&Tag was immediately conducted by following the manufacturer's instructions (EpiCypher). Nuclei from approximately 100000 sorted cells were separated, attached to concanavalin A coated magnetic beads, and then incubated with primary antibodies against RNA polymerase II CTD repeat YSPTSPS (Abcam), H3K4me3 (Active motif), H3K27ac (Abcam), or H3K36me3 (Abcam) at 4°C overnight. The next day, samples were washed and incubated with secondary antibodies and CUTANA pAG-Tn5 (EpiCypher). After washing, the tagmentation buffer (20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1x Roche cOmplete, EDTA-free Protease Inhibitor cocktail, and 10 mM MgCl2) was added to the tubes and rotated at 37°C for 1h. The samples were then incubated for at 58°C 1 h to release Cut&Tag fragment. Fragmented DNA was then amplified using Phusion High-Fidelity DNA polymerase (NEB) for 17-19 cycles with i5 & i7 sample index containing primers.