title: TGFB_S96_RH (DRIP-Seq)
design: For each sample, 10 ug of digested DNA was treated with 10 uL of RNaseH (New England BioLabs, M0297S) overnight at 37C. On Day 4, 10 ug of digested DNA not treated with RnaseH (two tubes per sample) as well as 10 ug of RNaseH-treated DNA was diluted in 500 uL of TE and 50 uL was stored for downstream qPCR applications. 52 L of 10x DRIP binding buffer (100 mM sodium phosphate, pH 7, 1.4 M NaCl and 0.5% (vol/vol) Triton X-100) and 20 L of the S9.6 antibody) were added to the remaining 450 L in each tube and incubated overnight at 4C in a mini-tube rotator. On Day 5, 40 uL of Pierce protein A/G magnetic beads slurry (ThermoFisher Scientific, 88803) per tube was washed with 700 uL of 1x DRIP binding by inverting the tubes on a mini-tube rotator for 10 min at room temperature. The beads were isolated using a DynaMag-2 magnet (ThermoFisher Scientific, 12321D) and supernatant was discarded. This wash step was repeated twice. The S9.6:gDNA solution was then added to the washed beads and incubated at 4C for 2 h while gently rotating on a mini-tube rotator. Next, the beads were isolated using a DynaMag-2 magnet and supernatant was discarded. 750 L of 1x DRIP binding buffer was added to each of the tubes which were then inverted for 15 mins at room temperature. The beads were isolated again and the wash step was repeated once more. Next, 300 L of DRIP elution buffer (50 mM Tris, pH 8.0, 10 mM EDTA, pH 8.0, and 0.5% (vol/ vol) SDS) and 7 L of proteinase K (20 mg/mL) was added to the beads and the tubes incubated at 55C for 45 mins, gently vortexing the tubes every 10 mins. The beads were isolated for the final time and the supernatant was transferred to pelleted 2 mL light phase-lock gel tubes and spun at 16,000g for 1 min. 300 L of phenol/chloroform/isoamyl alcohol (25:24:1) was added to each tube, which were gently inverted and spun at 16,000g for 10 mins at room temperature. The top layer was then transferred to a new 1.5 mL tube containing 1.5 L of glycogen, 25 L of 3M NaOAc, pH 5.2, and 625 uL of 100% ethanol, inverted 5 times and incubated at -20C for 1 h. Tubes were spun at 16,000g for 35 mins at 4C. The supernatant was discarded, the pellet was washed with 200 L of ethanol, and spun for 16,000g for 10 min at 4C. The pellet was airdried for 10-15 mins. Tubes containing samples pre-treated with RNaseH were resuspended in 50 uL of TE buffer and incubated on ice for 30 mins. Tubes containing samples not previously treated with RNaseH (2 tubes per sample) were resuspended in 25 uL of TE buffer, incubated on ice for 30 mins, and then combined into one 50 uL tube. qPCR was used to quantify pull-downs at various loci.