P-TABM-6290 - P-TABM-6290
Chromatin immunoprecipitation with TAP-tagged yeast strains: For the yeast TAP-tagged strains, ChIP was performed as described by (Aparicio et al., 2005), with modifications. Briefly, yeast strains containing TAP-tagged versions of the proteins as well as an untagged wild-type strain (for mock IP) were grown in 600 ml YPD medium to mid-log phase (OD600 ~ 0.8). Yeast cultures were treated with formaldehyde (1%, Sigma F1635) for 20 min at room temperature. Cross-linking was quenched with 75 ml of 3 M glycine for 30 min at room temperature. All subsequent steps were performed at 4 C with pre-cooled buffers and in the presence of a fresh protease-inhibitor mix (1 mM Leupetin, 2 mM Pepstatin A, 100 mM Phenylmethylsulfonyl fluoride, 280 mM Benzamidine). Cells were collected by centrifugation at 4000 rpm (Sorvall SLA-1500 rotor, Sorvall Evolution RC centrifuge) for 5 min, washed twice with 1x TBS (20 mM Tris-HCL at pH 7.5, 150 mM NaCl) and twice with FA lysis buffer (50 mM HEPES-KOH at pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na deoxycholate, 0.1% SDS, 1x protease inhibitor mix). Cell pellets were flash-frozen in liquid nitrogen and stored at -80 C. Cell pellets were thawed on ice and resuspended in 1 ml FA lysis buffer. Cells were disrupted by vortexing (neoLab 7-2020) in the presence of 1 ml silica-zirconia beads (Roth) for 3 min at full speed at 4 C, followed by an incubation of the sample for 2 min on ice. This was repeated 12 times. The success of the cell lysis was monitored by photometric measurements and the cell lysis efficiency was usually >80%. The chromatin was washed twice with FA lysis buffer and sonicated by application of a BioruptorTM UCD-200 (Diagenode Inc.) to yield an average DNA fragment size of 250 bp as determined by agarose gel electrophoresis. This was achieved by sonifying the sample 35 min at the ÔhighÕ intensity setting with alternating sessions of 30 sec of sonication followed by 30 sec of resting. 30 µl and 100 µl of the washed and fragmented chromatin samples were saved as input materials and for control of the average chromatin fragment size, respectively. 800 µl of the remaining chromatin sample was immunoprecipitated with 20 µl IgG SepharoseTM 6 Fast Flow beads (GE Healthcare) at 4 C for 4 h on a turning wheel. Immunoprecipitated chromatin was washed 3 times with FA lysis buffer, twice with high-salt FA lysis buffer (500 mM instead of 150 mM NaCl), twice with ChIP wash buffer (10 mM Tris-HCl at pH 8.0, 0.25 M LiCl, 1 mM EDTA, 0.5% NP-40, 0.5% Na deoxycholate) and one time with TE buffer (10 mM Tris-HCl at pH 7.4, 1 mM EDTA). Immunoprecipitated chromatin was eluted for 1 h at 65 °C in the presence of the ChIP elution buffer (50 mM Tris-HCl at pH 7.5, 10 mM EDTA, 1% SDS). Eluted immunoprecipitated chromatin as well as input material and material for control of the average chromatin fragment size were subjected to Proteinase K (20 µl of 20 mg/ml Proteinase K from Engyodontium album, Sigma P4850) digestion at 37°C for 2 h and reversal of crosslinks (at 65°C over-night). Samples used for determining the average chromatin fragment size were phenol-extracted twice and ethanol-precipitated over-night. The pellet was resuspended in 20 µl TE buffer (10 mM Tris-HCl at pH 7.4, 1 mM EDTA at pH 8.0) and incubated with 10 µl RNase A/T1 Mix (2 mg/ml RNase A, 5000 U/ml RNase T1; Fermentas) at 37°C for 1 h. The resulting DNA sample was electrophoretically separated on a 1.5% agarose gel. DNA of the IP, mock IP and input samples was purified with the QIAquick PCR Purification Kit (Qiagen) according to the manufacturer’s instructions, except that the final elution was performed with 100 µl DNAse-free water. RNA was digested by adding 5 µl of RNAse A (10 mg/ml, Sigma) at 37°C for 20 min. DNA was again purified with the QIAquick PCR Purification Kit (Qiagen) according to the manufacturer’s instructions. In case of the IP sample, the eluate was concentrated via vacuum manifold to a final volume of 10 µl. The total volume was used for DNA amplification (see below). Chromatin immunoprecipitation of Rpb3 in wildtype and Spt5?CTR background: For ChIP analysis of Rpb3 chromatin preparation was performed as above. For chromatin immunoprecipitation an antibody against Rpb3 (Neoclone) was used. 30 µl and 100 µl of the washed and fragmented chromatin samples were saved as input materials and as control of the average chromatin fragment size, respectively. The remaining 800 µl of sheared chromatin solution was immunoprecipitated with 5 µl Rpb3 mouse monoclonal antibody (Neoclone) at 4°C overnight on a rotating wheel, respectively. 25 µl of Protein A and Protein G Sepharose were added and incubated at 4°C for 1.5 h on a rotating wheel. Immunoprecipitated chromatin was treated as described above. DNA amplification: DNA samples were amplified and re-amplified with GenomePlex© Complete Whole Genome Amplification 2 (WGA2) Kit using the Farnham Lab WGA Protocol for ChIP-Chip (
). DNA quantity and quality control was performed with a ND-1000 Spectrophotometer (NanoDrop Technologies) and was usually larger than 1 µg. In addition, DNA quality was monitored by agarose gel electrophoresis. The re-amplification was performed in the presence of 0.4 mM dUTP (Promega U1191) to allow later enzymatic fragmentation.