GSM8887595: 02_T3BKO2; Mus musculus; OTHER
The Cut and Tag protocol was carried out following Zhang et al (80). Briefly, 2 ml 2XCB (0.1 M Tris pH 8.0; 0.3 M NaCl; 0.1% Triton X-100; 25% Glycerol), 2 ml pA-Tn5 tranposome (at 4 mM), and 2 ml S9.6 antibody were mixed in a microcentrifuge tube (mixture A) and kept on ice. One million cells were harvested and transferred to a clean 1.5 mL tube. Cells were washed once with 1 ml of PBS, suspend in 1 mL of 1X CB, and incubated on ice for 10 min. The cells were spun down, suspend in 1 mL of 1X CB. An aliquot of 50 ml was added to mixture A, incubated at room temperature for 60 min with rotation. 500 ml of wash buffer (50 mM Tris pH 8.0; 150 mM NaCl; 0.05% Triton X-100) was added to each tube, centrifuged. The pellets were washed two more time with 500 ml of wash buffer and then suspended in 100 ml of wash buffer. 1 ml of 1 M MgCl2 was added to start the transposome reaction, incubated at 37°C for 60 min. 4 ml of 0.5 M EDTA, 2 ml of 10% SDS and 1 ml of 20 mg/mL Proteinase K were added, and incubated at 55 °C for 60 min. The DNA was purified with ChIP DNA clean & concentrator (Zymo Research). Library was constructed following manufacture's protocol (Illumina) and sequenced on Next-seq. The kits used for the library prep were NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645) and NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs, NEB, E6440) were used with 13 cycles of PCR amplification. The final 750 pM library was sequenced paired-end with dual index (60x8x8x60) using the Illumina NextSeq2000 P2 Reagents (100cycles) on the Illumina NextSeq2000 instrument.