Whole genome recruitment of Rpb3 and Rpb4 in S.cerevesiae

ChIP on chip microarray experiment to decipher Rpb3 and Rpb4 recruitment in S.cerevesiae genome. Keywords: ChIP-chip TAP- tagged Rpb3 and Rpb4 strains were cultured in 100 ml YPD medium at 25°C till log phase (O. D. 600 ~ 0.8). Cells were crosslinked with formaldehyde to a final concentration of 1% for 20 minutes. Cells were collected by centrifugation, washed three times with 40 ml of ice-cold Tris-buffered saline buffer (20 mM Tris HCl [pH 7.5], 150 mM NaCl), and resuspended in 800ul of lysis buffer (50 mM HEPES-KOH [pH 7.5], 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, Protease inhibitor cocktail supplied by Roche). Yeast cells were disrupted by shaking for 30 sec for 20 times in the presence of glass beads (diameter, 0.5 mm) using vortexer. Glass beads were removed, and samples were sonicated five times for 15 sec at 20% duty cycle on a sonicator in order to shear chromatin DNA into fragments of average size ~400- 500 bp. Samples were centrifuged 10 min at maximum speed in a micro centrifuge, and the supernatant (from now on referred to as whole-cell extract) was used as input material for immunoprecipitation. Two hundred microliters of whole-cell extract was incubated with Rabbit IgG agarose beads (Sigma) for 3 hrs at 4°C with agitation. Beads were washed twice with 1 ml of lysis buffer, twice with 1 ml of lysis buffer plus 500 mM NaCl, twice with 1 ml of wash buffer (10 mM Tris-HCl [pH 8.0], 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, and 1 mM EDTA) and once with 1 ml of Tris-EDTA (TE; 10 mM Tris-HCl [pH 8.0], 1 mM EDTA). Bound material was eluted from the beads by resuspending beads in 100ul of elution buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, and 1% SDS) and incubating 10 min at 65°C with occasional agitation. Samples were centrifuged briefly and the cross-linking was reversed by incubating overnight at 65°C. Samples were treated with proteinase K and RNase A, extracted twice with phenol, extracted once with chloroform, precipitated with ethanol, and resuspended in 30ul of TE. These samples were further purified with Qiagen PCR product purification Kit.