Illumina HiSeq 2500 sequencing; Glucocorticoid and androgen receptor profiling (ChIP-seq) in U2OS-GR and U2OS-AR cells upon hormone treatment
Glucocorticoid and androgen receptor profiling (ChIP-seq) in U2OS-GR and U2OS-AR cells upon hormone treatment
ChIP-seq was performed on 5 million cells per treatment. Cells were cross-linked with 1% formaldehyde for 3 min and harvested. Cells where grown to approximately 90% confluence according to standard protocols in DMEM medium with 5% FBS. U2OS-GR cells were treated with 1 µM dexamethasone for 90 minutes. U2OS-AR cells were treated with 5 nM R1881 for 4 hours. Cells 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). Antibodies used: GR, N499 (2 µl/ChIP); AR, polyclonal antibody PG-21 (Anti-AR; Sigma Aldrich; 06-680, 2 µl/ChIP) Sequencing libraries were prepared using the NEBNext Ultra DNA Library Prep kit (NEB E7370) according to manufacturer's instructions.