GSM2300993: B6_thymocytes_ZFAT_ChIPseq; Mus musculus; ChIP-Seq
Thymocytes (1.5 × 10^8 cells) from 4 to 5-weeks-old C57BL/6, Zfatf/f or Zfatf/f-LckCre mice were crosslinked with 1% formaldehyde for 10 min at room temperature. Crosslinking reaction was quenched with 125 mM glycine for 5 min. The cells were rinsed with PBS twice and incubated in 600 µl of MNase treated buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% NP-40, 5 mM CaCl2) containing 375 unit MNase (Roche) for 0.5 h at 37°C. The MNase reaction was stopped by adding 600 µl of lysis buffer  (100 mM Tris-HCl, pH 8.0, 20 mM EDTA, 2% SDS, protease inhibitor cocktail (Roche)), and the lysate was sonicated using a Bioruptor (Cosmo Bio, Tokyo, Japan) for two cycles of one minute with 30 seconds on/off. The sonicated lysate was subjected to immunoprecipitation with a monoclonal antibody against ZFAT (prepared in Dr. Shirasawa's lab), H3K9me3 (MBL), or H3K9ac/K27ac (MBL). The immunoprecipitated DNA was purified using MinElute Reaction Cleanup kit (Qiagen) according to the manufacturer’s instructions. ChIP-seq libraries were prepared using NEBNext ChIP-seq Library Prep Master Mix Set and Multiplex Oligos for Illumina (New England Biolabs) according to manufacturer's instructions. ChIP or input DNA (0.1 ~ 1 ng) was subjected to end repair, dA-tailing, and adaptor-ligation, and amplified by 6-13 cycles of PCR.