RBPJ Chromatin Immunoprecipitation Protocol

5–10×10⁶ RD cells were plated as spheres for 24–48 hours. Spheres were then cross-linked with 1% formaldehyde for 10–15 min, quenched with 0.125M glycine for 10min, and washed with PBS. Cells were resuspended in lysis buffer (50mM Tris pH7.5, 150mM NaCl, 5mM EDTA, 1% Triton-X, 0.1% SDS, 0.5% sodium deoxycholate) and sonicated (Misonix XL-2000) for 14 cycles (12 sec on, 2 min off). Cell debris was pelleted, and chromatin was precleared with Protein G agarose beads (Millipore) for 2 hours at 4°C. RBPJ antibody (Cell Signaling #5313) was added at 1:50 and rotated overnight at 4 °C. Protein G beads were added the next day for 3 hours with rotation at 4 °C. Beads were washed according to the Abcam protocol and DNA was eluted with elution buffer (Santa Cruz) at 67°C for 2 hours with rotation. Crosslinks were reversed overnight followed by a proteinase K digestion for 1hr. DNA was purified using the Qiagen PCR Purification kit. ChIP enrichment was evaluated using semi-quantitative PCR followed by quantitation using ImageJ (NIH) similar to previous published work (57 (link)). ChIP primers are listed in Supplementary Table 1.