GSM8005118: GR_ChIP_dKO_VEH_BioRep2; Mus musculus; ChIP-Seq
For chromatin immunoprecipitation, livers were minced in PBS containing 1% formaldehyde and rotated end-over-end at room temperature for 20 minutes. Samples were then quenched with glycine, washed with PBS, and dounce homogenized in ChIP buffer (50 mM HEPES pH 7.4, 1.1% Triton X-100, 0.11% Sodium Deoxycholate, 1 mM EDTA, 155 mM NaCl, supplemented with protease and phosphatase inhibitors), and sonicated via probe sonication (Branson). Homogenates were subsequently subject to chromatin immunoprecipitation via overnight incubation with the following antibodies: NCOR1 (Generated in-house and previously described, raised in rabbit against amino acids 1944-2453), IgG (Cell Signaling 2729), GR (2 ug each of: Santa Cruz-393232, Thermo Fisher MA1510, Thermo Fisher PA1-511A). All antibodies have been previously validated in the literature for ChIP. ChIP conjugates were captured via Protein-A/G sepharose beads blocked with BSA, exhaustively washed, eluted, reverse crosslinked, and subject to protease digestion followed by DNA isolation. ChIP libraries were generated using the NEBNext Ulta II DNA library Prep Kit (NEB). Library size and quality was assessed using DNA highsensitivity kits on an Agilent Tapestation and sequenced on a NOVAseqX Plus.