GSM7910307: SMC2_noc_with_auxin_rep3_hp1b; Mus musculus; ChIP-Seq
FACS purified cells were re-suspended in 1ml pre-cooled Cell Lysis Buffer supplemented with 1:500 protease inhibitor and 1:100 PMSF for 20min on ice. To control for global change of histone modification that cannot be detected by ChIP-seq, HEK293T cells were used as spike-in controls and added into FACS purified mitotic and asynchronous cells so that both samples contain comparable level of human genome (~10%). Nuclei were then re-suspended in 1ml Nuclear Lysis Buffer (50mM Tris pH 8, 10mM EDTA, 1% SDS, fresh supplemented with PI and PMSF) for 20min on ice. Chromatin was then fragmented to 200-300bp by sonication (Qsonica Q800R3, 80% amplitude, 20s ON, 40s OFF) for 17min. Samples were centrifuged at 15000g for 10min at 4℃ to remove cell debris. Chromatin in the supernatant was collected and diluted with 4 volumes of IP dilution buffer (20mM Tris pH 8.0, 2mM EDTA, 150mM NaCl, 1% Triton X-100, 0.01% SDS) and then supplemented with protease inhibitor, PMSF and 50ul of A/G agarose beads (Santa Cruz Cat#sc-2003). Preclear of chromatin was performed at 4℃ for 8h with rotation. Precleared chromatin was then incubated with 35μl A/G agarose beads (A:G = 1:1) pre-bound with ChIP antibody at 4℃ overnight. Beads were washed once with IP wash buffer I (20mM Tris pH 8, 2mM EDTA, 50mMNaCl, 1% Triton X-100, 0.1% SDS), twice with high salt buffer (20mMTris pH 8, 2mM EDTA, 500mMNaCl, 1% Triton X-100, 0.01% SDS), once with IP wash buffer II (10mMTris pH 8, 1mM EDTA, 0.25 M LiCl, 1% NP-40/Igepal, 1% sodium deoxycholate) and twice with TE buffer (10mM Tris pH 8, 1mM EDTA pH 8). All washing steps were performed on ice. Beads were moved to room temperature and DNA was eluted in 200μl fresh Elution Buffer (100mM NaHCO3, 1%SDS). ChIP and input chromatin were reverse crosslinked at 65℃ overnight in the prescence of proteinase K.  IP and input samples were supplemented with 10ul of 3M sodium acetate (pH 5.2), and DNA was purified with QIAquick PCR purification kit (QIAGEN Cat#28106). ChIP-seq libraries were constructed using the AHTS Universal DNA Library Prep Kit for MGI (Vazyme, Cat#NDM607-02) according to the manufacture's protocol.For cut and tag library: we performed CUT&TAG on asynchronously growing G1E-ER4 cells using lamin B1 antibody (ab16048) according to the manufacture's protocol (Vazyme Hyperactive® Universal CUT&Tag Assay Kit for Illumina).