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. Antibody: H3K27ac (Active Motif, 39135) Cells grown in 2iLIF on gelatin coated plates and transferred to SerumLIF medium on gelatin for 48h 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: SAMEA7195772
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:WT_H3K27ac_2_SL
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: TNG-A:TbC1
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: genotype
attributes.value: wild type genotype
attributes.tag: growth condition
attributes.value: SL
attributes.tag: sample name
attributes.value: E-MTAB-9450:WT_H3K27ac_2_SL
attributes.tag: scientific_name
attributes.value: Mus musculus