Ab4174

In total, 2 × 10⁷ MCF7 cells transfected with control or H2A.Z siRNA were washed once with cold PBS and then lysed with PBS containing 0.5 % TritonX-100, 2 mM PMSF, 5 mM sodium butyrate, and protease inhibitors. To extract histones, the pellet was further incubated with 0.4 M H₂SO₄ for 2 h at 4 °C.
The extracted protein was separated on a 4–15 % SDS-PAGE gel, transferred to a PDVF membrane, and probed with H2A.Z antibody (ab4174, Abcam) or Histone H3 antibody (ab1791, Abcam) to detect protein expression.

Hundred to two hudredred milligrams of flash-frozen liver tissue was homogenized with a tissue Dounce as previously described and subsequent micrococcal digest, chromatin immunoprecipitation, and library preparation were carried out as described^{2 (link)}. The antibodies used for ChIPseq and the concentrations, volumes and sources are anti-H3K4me3 (Abcam, ab1012 1 µg/µl; used 6 µl), anti-H3K27me3 (Active Motif, 61017; 1 µg/µl; used 6 µl), anti-H3K9me3 (Active Motif, 39161; 1 µg/µl; used 6 µl), and anti-H2AZ (Abcam, ab4174; 1 µg/µl; used 10 µl). The prepared libraries were sequenced on the Hiseq2500 platform for 75 bp single-end or 100 bp paired-end reads read runs at the Core Technology Platforms (CTP) at New York University Abu Dhabi (NYUAD). After sequencing, 75 base-pair single-end or 100 base-pair paired-end reads that passed quality trimming were aligned against the mouse reference genome (GRCm38.p4) using BWA-MEM^{77 (link)}. The resulting BAM files were then processed through PICARD tools (to clean, deduplicate and sort) for downstream analysis and visualization. One sample from each timepoint and antibody was used.

The proteins extracted above were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Briefly, 12% gel in SDS-PAGE was used to separate the proteins with different molecular weight and then all the proteins were electrotransferred onto a polyvinylidene difluoride (PVDF) membrane using a Bio-Rad Trans-Blot system. 5% skim milk powder was used to block the membranes in TBST buffer (20 mM Tris–HCl, 150 mM NaCl, 0.05% Tween 20) for 30–60 min at room temperature. After that, the PVDF membranes were incubated with pan anti-acetyl lysine antibody (PTM Biolabs, 101), anti-histone H2A.Z antibody (Abcam, ab4174) and anti-H2A.Z (acetyl K7) antibody (Abcam, ab214730) (1:1000, in TBS/5% skim milk powder) for 1 h at room temperature, respectively. After the PVDF membranes were washed 3 times with TBST buffer, the membranes were incubated with horseradish peroxidase (HRP) conjugated anti-rabbit IgG (1:5000 dilutions) for 1 h at room temperature. Last, an enhanced chemiluminescence (ECL) immunoblotting detection kit (Beyotime Biotechnology, Shanghai) was used for signal detection.

Total Cell extracts were prepared and analysed by standard Western blotting protocol, using antibodies against total H2AZ (ab4174, Abcam), GAPDH (clone 6C5, MAB374, Millipore), PHF14 (SAB3500960, Sigma and proteintech 24787–1-AP), SIRT1 (Clone E104, ab32441, Abcam), Flag (Clone M2, F1804, Sigma), Pol I (Clone RPA194, sc48385, Santa Cruz), H3 (ab1791, Abcam), pan Acetyl-H3 (6599, Upstate), Acetyl-H3K9 (06942, Upstate), Flag-HRP (Sigma, A8592, lot #GR08726011-2013), Brd8 (Bethyl A300-219A), DMAP1 (Aff. BioReag., PA1-886), ACTR6(ARP6) (Abcam, ab208830), p400 (Abcam, ab5201), SRCAP (gift from J. Chrivia), BAF53a (Abcam, ab3882, lot #9118237), EPC1 (Abcam, ab5514, lot#98723).

The expression vector containing the PA-Tnp fusion construct is available from Addgene under accession number 121137 [http://www.addgene.org/121137/]. Recombinant PA-Tnp protein was expressed in BL21-Gold(DE3) competent cells (Agilent cat # 230132) and purified using nickel beads. Quality and purity of the isolated protein was assayed using SDS-PAGE. In all, 10 μM of recombinant PA-Tnp protein was incubated for 10 min at 25 °C in complex formation buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.05% Triton X-100, 12.5% glycerol) containing 50 μM of 5′ complex barcode and 50 μM of 3′ complex barcode. 1 μL of the appropriate antibody (at the stock concentration provided by the manufacturers) was added to 12 μL of prepared complex and incubated at 25 °C for 60 min. The antibodies used in this study, listed with the manufacturer’s provided concentration information, were: anti-H3K4me3 (Millipore cat # 17-614, indicated 3 μL for one ChIP sample), anti-H3K4me2 (Abcam cat # ab32356, 0.1–0.5 mg/mL), anti-H3K4me1 (Abcam ab8895, <1 mg/mL), anti-H3K27ac (Abcam ab4729, <1 mg/mL), anti-H2A.Z (Abcam ab4174, 0.8 to 1 mg/mL), anti-BRD4 (Bethyl cat # A301-985A100, 1 mg/mL), and normal IgGs (Millipore cat # cs200581, 1 mg/mL).