3 protocols
AccessionNameType
P-TABM-6291
labeling
DNA labeling and microarray handling: 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 (http://www.genomecenter.ucdavis.edu/farnham/protocol.html). 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. The enzymatic fragmentation, labelling, hybridization and array scanning were done according to the manufacturer’s instructions (Affymetrix Chromatin Immunoprecipitation Assay Protocol P/N 702238). Enzymatic fragmentation and terminal labelling were performed by application of the GeneChip WT Double-Stranded DNA Terminal Labeling Kit (P/N 900812, Affymetrix). Briefly, re-amplified DNA was fragmented in the presence of 1.5 µl uracil-DNA-glycosylase (10 U/µl) and 2.25 µl APE1 (100 U/µl) at 30°C for 1 h 15 min. The average fragment size was in the range of 50-70 bases as determined by automated gel electrophoresis on an Experion system (Bio-Rad Laboratories, Inc.) that allowed the analysis of small amounts of DNA. The fragmented DNA was then labelled at the 3’-end by adding 2 µl and 1 µl of terminal nucleotidyl transferase (TdT, 30 U/µl) and GeneChip DNA Labeling Reagent (5 mM), respectively. 5.5 µg of fragmented and labelled DNA were hybridized to a high-density custom-made Affymetrix tiling array (PN 520055) at 45°C for 16 h with constant rotational mixing at 60 rpm in a GeneChip Hybridization Oven 640 (Affymetrix, SantaClara, CA). Washing and staining of the tiling arrays were performed using the FS450_0001 script of the Affymetrix GeneChip Fluidics Station 450. The arrays were scanned using an Affymetrix GeneChip Scanner 3000 7G.
P-TABM-6290
immunoprecipitate
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 (http://www.genomecenter.ucdavis.edu/farnham/protocol.html). 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.
P-TABM-6289
grow
Yeast strains and epitope tagging: Saccharomyces cerevisiae (S. cerevisiae) BY4741 (MATa his3?1 leu2?0 met15?0 ura3?0) strains containing C-terminally tandem affinity purification (TAP) tagged versions of target proteins were obtained from Open Biosystems. The Spt5?CTR strain lacking 15 C-terminal hexapeptide repeats, amino acids 931 to 1063, of Spt5 was generated as described. BY4741 untagged wild-type strain (Open Biosystems) was used for mock IP in ChIP-Chip experiments. All epitope-tagged strains were validated. First, gene-specific PCR was performed to confirm that the TAP tag was at the correct genomic position. Second, Western blotting with anti-TAP (PAP, Sigma) antibodies was performed to verify if the tagged protein of interest was properly expressed. Third, the growth of the various tagged strains compared to the non-tagged wild-type strain was monitored to rule out any influence of the epitope tag on yeast growth. This was done by serial dilutions of the various yeast strains on YPD plates at 30 C for two days.