organism: Mus musculus
description: Protocols: 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 TNG-A mouse embryonic stem cells stabily expressing Cas9, transfected with gRNAs AGTTCTGCTCCCGTTACAC and GAGTCAATGCCAGGCGATG, 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)
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2021-03-23T08:56:58Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2020-08-19T12:34:23Z
attributes.tag: External Id
attributes.value: SAMEA7195767
attributes.tag: INSDC center name
attributes.value: Friedrich Miescher Institute for Biomedical Research
attributes.tag: INSDC first public
attributes.value: 2021-03-23T08:56:58Z
attributes.tag: INSDC last update
attributes.value: 2020-08-19T12:34:23Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-9450:13C_Input_2iLIF
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: TNG-A:13C
attributes.tag: cell type
attributes.value: mouse embryonic stem cell
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: embryo stage
attributes.tag: genetic modification
attributes.value: gene knock out
attributes.tag: genotype
attributes.value: HDAC3 -/-
attributes.tag: growth condition
attributes.value: 2iL
attributes.tag: sample name
attributes.value: E-MTAB-9450:13C_Input_2iLIF
attributes.tag: scientific_name
attributes.value: Mus musculus