GSM8988230: CT_Skp1aPcgf1cont_mb_EED_lot1; Mus musculus; OTHER
Cells were washed with DIG wash buffer (wash buffer with 0.05 % digitonin) and treat with secondary antibody for 1 hour at room temperature, then wash with Dig-300 buffer (20 mM HEPES pH7.5, 300 mM NaCl, 0.5 mM spermidine, 0.01 % digitonin). Samples were incubated with pA-Tn5 (Diagenode: C01070001) for one hour at room temperature and wash with Dig-300 wash buffer. Samples were suspended with 100 ul of tagmentation buffer (Dig-300 buffer with 10mM MgCl2) and Incubate at 37°C for 30 minutes. Then, stop the reaction with 3.3 ul of 500 mM EDTA, 1 ul of 10% SDS, and 1 ul of 20 mg/ml Proteinase K, and then incubate at 65°C for 6 hours and 55°C overnight. DNA from these samples was purified using SPRI beads, and eluted DNA samples were used as 3D samples. Eluted samples were used for the CUT&Tag library. CUT&Tag libraries were prepared by PCR with Nextera primers as described in Kaya-Okur and Henikoff (dx.doi.org/10.17504/protocols.io.wnufdew).