GSM8387236: mESC, Nsd1, Flag ChIP; Mus musculus; ChIP-Seq
E14 single cell suspension was subject to a dual cross-linking protocol: initial fixation under 2mM DSG (Thermo Fisher: 20593) in PBS for 45min at RT, followed by fixation in 1% formaldehyde in PBS (Thermo Fisher: 28908) for 10min, and then quenched by addition of 125mM glycine for 5min. The cross-linked cells were sequentially lysed or washed in LB1, LB2, and LB3, followed by sonication for 60 cycles on Bioruptor sonicator at high energy with 30s on and 30s off (Diagenode: B01020001). The cleared sonication supernatant was then incubated with Flag (Sigma: F1804) or HA (abcam: ab9110) antibody-bound dynabeads (Thermo Fisher: 11202D,11204D) overnight at 4 °C. After sequential washes with low-salt buffer, high-salt buffer, LiCl buffer, and TE buffer, the ChIP product was eluted and subject to overnight reverse crosslink at 65 °C, followed by RNase (Roche: 11119915001) and Protease K (Roche: 03115828001) digestion, and DNA recovery by Qiagen PCR purification kit (Qiagen: 28106). The ChIP-seq libraries were generated using NEBNext Ultra II DNA Library Prep Kit (NEB: E7645L)