CtrlG_FB_DRIP_S96
"DNA-RNA Immunoprecipitation (DRIP) procedure was adapted from previous studies (Skourti-Stathaki et al., 2011).  5X106 primary fibroblasts were lysed in 600ul cell lysis buffer (50mM PIPES, pH 8.0, 100mM KCl, 0.5% NP-40) and nuclei were collected by centrifugation.  Nuclei pellet was resuspended in 300ul nuclear lysis buffer (25 mM Tris-HCl, pH 8.0, 1% SDS, 5mM EDTA).  Genomic DNA along with R-loops was then extracted by phenol:chrolorform and precipitated by ethanol.  Purified DNA was resuspended in IP dilution buffer (16.7mM Tris-HCl, pH 8.0, 1mM EDTA, 0.01% SDS, 1% Triton-X100, 167mM NaCl) and sonicated 15 minutes in Bioruptor (Hi setting, 30sec On/30sec Off) to fragments with average size of 500bp.  3ug of S9.6 monoclonal antibody (gift from Dr. Stephen H. Leppla at NIH) or non-specific mouse IgG was used for each immunoprecipitation.  Sequencing libraries were prepared from input and DRIP DNA using Ovation Ultralow System (Nugen) and sequenced on Illumina HiSeq 2500 platform.  An average of 100 million 100-nt reads per sample were generated."