Nuclei isolating kit

Nuclei were purified from cells according to the manufacturer's protocol with Nuclei Isolating Kit (NUC101-1KT, Sigma-Aldrich). Then nuclei were resuspended with DNase I digestion buffer and treated with indicated units of DNase I (Sigma-Aldrich) at 37°C for 5 min. 2 × DNase I stop buffer (20 mM Tris Ph 8.0, 4 mM EDTA, 2 mM EGTA) was added to stop reactions. DNA was extracted and examined by PCR.

Nuclei were isolated from mouse BM cells using the Nuclei isolating Kit (NUC101, Sigma-Aldrich) according to the manufacturer’s protocol, followed by micrococool MNase digestion. Gel filtration chromatography was performed by Superdex 200 10/300 GL (GE Healthcare) according to the manufacturer’s instruction to isolate stripped nucleosome fractions. SRCAP was immunoprecipitated by anti-SRCAP antibody and protein A/G beads from nuclear extracts of Pcid2^+/+ or Pcid2^−/− MPPs. His-tagged H2A.Z and H2B protein were purified from E. coli and assembled to H2A.Z/H2B dimer in vitro. Purified His-tagged PCID2 or GST-tagged ZNHIT1 protein (4 μg for each protein) was directly added into the exchange reaction buffer (70 mM NaCl, 10 mM Tris-HCl (pH8.0), 5 mM MgCl₂, 0.1 mg/ml BSA, 2 mM ATP and 1 mM DTT). Isolated mononucleosomes, immunoprecipitated SRCAP complex, purified proteins of PCID2 or ZNHIT1 and recombinant His-H2A.Z-H2B dimers were incubated at 37 °C for 30 min and resolved by native gel. His-tagged H2A.Z exchanging into nucleosomes was probed by immunoblotting^{59 (link)}.

DNaseI digestion assay has been described previously^{28 (link)}. In brief, Nuclei were purified from CHILPs according to the manufacturer’s protocol with the Nuclei isolating Kit (Sigma-Aldrich). Then nuclei were resuspended with DNase I digestion buffer and treated with indicated units of DNase I (Sigma, USA) at 37 °C for 5 min. In all, 2 × DNase I stop buffer (20 mM Tris Ph 8.0, 4 mM EDTA, 2 mM EGTA) was added to stop reactions. DNA was extracted and examined by qPCR.

SSEA-1⁺ iPSCs were sorted by flow cytometry. Nuclei were isolated from 1 × 10⁵ iPSC cells using the nuclei isolating kit (Sigma-Aldrich) according to the manufacturer’s protocol. Nuclei were resuspended in 200 μl of DNase I digestion buffer (1 mM EDTA, 0.1 mM EGTA, 5% sucrose, 1 mM MgCl₂, and 0.5 mM CaCl₂). Equal aliquots of 100 μl of nuclei were treated with the indicated units of DNase I (Sigma, USA) and incubated at 37 ℃ for 5 min. The reactions were stopped by 2 × DNase I stop buffer (20 mM Tris, pH 8.0, 4 mM EDTA, and 2 mM EGTA). DNA was extracted and analyzed by qPCR. The primer pairs aimed at the promoter region for indicated genes are listed below. mNanog promoter: Forward, 5′-ATCCACCTGCCTCTGCCGCCTAA-3′, Reverse, 5′- GCATTGGTGTTTTGCCTGCATGG-3′; mEsrrb promoter: Forward, 5′-CCAACCAGAAGTGGGTCTTGTTCCT-3′, Reverse, 5′-TGTGGAAGGATCCTGGGACACAGAT-3′^{64 (link)}.