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. Cells grown in 2iLIF on gelatin coated plates and transferred to SerumLIF medium on gelatin for 48h TNG-A mouse embryonic stem cells stabily expressing Cas9, transfected with gRNAs AGTTCTGCTCCCGTTACAC and TGAAACCCTGCATATTGGT, mutant clone 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)