GR_ChIP-seq_dex_washout
Protocols: ChIP-seq was performed on 30 million cells. Cells were cross-linked with 1% formaldehyde for 3 minutes and harvested. Cells where grown to approximately 90% confluence according to standard protocols in DMEM medium with 5% FBS. Cells were treated with 100 nM dexamethasone for 4 hours, washed 2x with PBS and cultured for 24 hours in hormone-free medium before harvest. Cells were lysed with IP lysis buffer (50 mM HEPES-KOH pH 7.4, 1mM EDTA, 150 mM NaCl, 10% Glycerol, 0.5 % Triton X-100, 1:200 Protease inhibitor, 1:200 PMSF). After centrifugation pellets were resuspended in RIPA buffer (10 mM Tris-HCL, pH 8, 1 mM EDTA, 150 mM NaCl, 5 % Glycerol, 0.1 % DOC, 0.1 % SDS, 1 % Triton-X100, 1:200 Protease inhibitor, 1:200 PMSF) and sonicated with Diagenode Bioruptur using 24 cycles (30 sec on, 30 sec off) at 4 °C. After centrifugation lysate was resuspended in RIPA buffer. The antibody was added and incubated overnight. A 50% agarose bead slurry (Santa Cruz protein A/G agarose beads) was washed 2x with RIPA buffer and nutated overnight at 4 °C. RIPA buffer was removed and beads were added to the samples and incubated rotating for 4h at 4 °C. The beads were washed 5x with 1 ml RIPA wash buffer (RIPA containing 500 mM NaCl). ChIP samples were supplemented with Proteinase K solution (TE buffer pH 8, 0.7 % SDS, 200 µg/ml proteinase K) and incubated for at 55 °C for 3 h and 65 °C overnight. Next day 1 µl RnaseA (2 µg/ml) was added and incubated at 25 °C for 1 h. The ChIP samples were purified using the PCR Purification Kit (Promega, Wizard SVGel and PCR Clean-Up system). Antibody used: GR, N499 (2 µl/5 million cells) Sequencing libraries were prepared using the Kappa HyperPrep Kit (Roche 07962363001) according to manufacturer's instructions.
ENA first public
2021-05-12
ENA last update
2021-05-12
External Id
SAMEA7722233
INSDC center name
MPIMG
INSDC first public
2021-05-03T00:22:16Z
INSDC last update
2020-12-15T10:39:43Z
INSDC status
public
Submitter Id
E-MTAB-9914:GR_ChIP-seq_dex_washout
broker name
ArrayExpress
cell line
U2OS-GR
common name
human
developmental stage
adult
disease
osteosarcoma
organism part
bone
sample name
E-MTAB-9914:GR_ChIP-seq_dex_washout
stimulus
compound and washout