Illumina HiSeq 2500 sequencing; ChIPseq for histone marks in WT and Hdac3 knock-out clones of TNG-A derived mESCs in 2iLIF and SL differentiation conditions
ChIPseq for histone marks in WT and Hdac3 knock-out clones of TNG-A derived mESCs in 2iLIF and SL differentiation conditions
Native histone ChIP: 10 million cells were harvested and nuclei isolated by hypotonic buffer (10mM TrisHCl pH7.5, 10mM KCl, 1mM dithiothreitol (DTT), 0.5% IGEPAL) for 20' on ice. Nuclei were lysed by rotation for 90' at 4°C in 1mL of lysis buffer (20mM TrisHCl pH7.5, 100mM KCl, 1.5mM MgCl2, 1mM DTT, 10% glycerol, 1x Protease Inhibitor Tablet (Roche), 2x Phosphatase Inhibitor Tablets (Roche), 0.5% Triton X-100, Benzonase 250 units/mL). 10% of material was set aside as input. Clarified lysates were incubated with 4µg of primary antibody for 2h and then SDS concentration was brought to 0.1% and EDTA to 5mM to inactivate benzonase both for ChIPs and input samples. Lysates were clarified again and 40µL of protein G Dynabeads were added and rotated for 6h at 4°C. Input samples were frozen and beads were washed twice with RIPA buffer, four times with RIPA buffer with 500mM NaCl, and once with TE. Antibody: H3K27ac (Active Motif, 39135) Cells grown in 2iLIF on gelatin coated plates DNA was extracted by proteinase K treatment in 0.5% SDS for 3h at 55°C and purified with Qiagen MinElute PCR Purification Kit. ChIP-Seq NEB Ultra (Dual Indexes)