anti-TBP 517 Antibody (custom polyclonal antibody generated in rabbits)
Sequenced DNA Library
ChIPs were performed with whole cell extracts (WCE) from embryos as previously described (Chen et al. 2013) with small modification. 800 mg 2-4h embryos were crosslinked with 1.8% Formaldehyde in 2.4 ml crosslinking buffer with 7.5 ml Heptane for 15 min. After wash with PBT-0.125M glycine twice, embryos were transferred to a 7 ml Douncer homogenizer with 5 ml Buffer A1 (15 mM HEPES, pH 8; 15 mM NaCl; 60 mM KCl; 4 mM MgCl2; 0.5% Triton X-100; 0.5 mM DTT; fresh 1 × Protease Inhibitor Cocktail), and homogenized five times each with the loose fitting and the tight fitting pestles. Homogenized samples were centrifuged 3 min at 4°C at 1500 g, and the pellet was then subsequently washed three times with Buffer A1 and once with Buffer A2 (15 mM HEPES, pH 8; 140 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 1% Triton X-100; 0.1% sodium deoxycholate; 1% SDS; 0.5% N-lauroylsarcosine; fresh 1 × Protease Inhibitor Cocktail). Resuspended the pellet with 2.8 ml Buffer A2 and aliquot to 4 EP tubes. Sheared the chromatin with Branson Sonicator 8 × 10s at Power 3. Sheared chromatin was cleaned by centrifuge 10 min at 4°C at 13000 g. Supernatant was collected for ChIP. Aliquot 130ul Rabbit IgG Dynabeads to 4 tubes and wash with PBS-0.5%BSA twice. Incubated beads with antibodies (10ul for TBP and 30 ul for TRF2) in 700ul PBS-0.5%BSA for 2 h at 4°C. removed PBS-0.5%BSA, added 700 ul sheared chromatin for each tube, and incubated overnight at 4°C. the ChIPed chromatin was washed with RIPA buffer 3 times, and eluted with 200 ul elution buffer + 200ul TE. Incubated the chromatin 30 min at 37°C with RNase (60ug) and 6h at 65°C for reversing the crosslink with Proteinase K (60ug). DNA was clean by phenol/phenol-chloroform-isoamylalcohol extractions and ethanol precipitation. The precipitated DNA was resuspended in 35 μl water. Libraries for the ChIPed DNA were prepared and barcoded with NEBNext DNA Library Prep Master Mix and Multiplex Oligos for Illumina. For each step, DNA was cleaned with Ampure beads as suggested in the NEB manual.