biomaterial: Saccharomyces cerevisiae BQS252. Exponential growth in YPD
Growth protocol
Exponential growth in YPD
Extracted molecule
genomic DNA
Extraction protocol
50 mL of cells were cross-linked by adding formaldehyde to a final concentration of 1% for 15 minutes at room temperature, and the cross-link was quenched by adding glycine to a final concentration of 125 mM. Cell were washed 4 times with 30 mL ice-cold TBS buffer (20mM Tris-HCl, 140 mM NaCl, pH 7.5) and frozen. Cells were thawed on ice and resuspended in 300 μl lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 1 mM PMSF (Phenylmethylsulfonyl fluoride), 1 mM benzamidine and 1 pill of protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) dissolved in every 50 ml of buffer). The equivalent of 0.3 ml of frozen glass beads (425-600 mm; Sigma-Aldrich, St Louis, MO, USA) was added to the cellular suspension and cells were lysed at 4°C for 12 minutes of vortexing at maximum power. Lysis buffer (300 μl) was added and the extract was transferred to a new tube. Sonication of DNA was performed to obtain DNA fragments between 200 and 500 bp, with an average size of 350 bp. Chromatin was sonicated on ice-cold water, with 5 pulses of 30 s at High output (200W) in a Bioruptor (Diagenode SA, Liège, Belgium). Cell debris was removed by centrifugation at 12,000 rpm at 4°C for 5 minutes. An aliquot of 10 μl of this whole cell extract (WCE) was kept as positive control.
Label
33P
Label protocol
Ligation Mediated PCR (Ren B. et al. 2000) was used for DNA amplification. Briefly,the DNA was blunted by T4 phage DNA polymerase. The reaction was allowed to proceed for 20 minutes at 12°C. After phenol/chloroform/isoamylic alcohol extraction, DNA was ethanol-precipitated and ligated in a final volume of 50 μl with annealed linkers oJW102 and oJW103 (1.5 μM of each primer). The reaction was carried out overnight at 16°C and ligated DNA was precipitated and resuspended in 25 μl milliQ water. The ligated DNA was amplified by PCR. The PCR program was 2 minutes at 95°C and 33 cycles of 30 s at 95°C, 30 s at 55°C and 2 minutes at 72°C. The DNA was precipitated overnight and resuspended in 50 μl of milliQ water. The LMPCR product was used as a template for the radioactive labeling reaction. The reaction mixture contained 5-15 μL of amplified DNA in 50 μl (1× DNApol buffer, 2 mM MgCl2, 0.2 mM dATP, dTTP and dGTP, 25 μM dCTP, 1 μM oJW102, 0.8 μCi α-33P-dCTP and 5 U DNApol). The mix was denatured for 5 minutes at 95°C, annealed 5 minutes at 50°C and was amplified during 30 minutes at 72°C. The reaction product was purified through a ProbeQuant G-50 column to remove unincorporated 33P-dCTP and oligonucleotides.
Hybridization protocol
Hybridization Solution was: 5X SSC, 5X Denhardt’s, 0.5% SDS and 100 µg herring sperm DNA/ml. The hybridization protocol used was as follows. Filters were inserted in 12.5X 2.5-cm flat-bottom plastic tubes and pre-hybridized in a rotator oven with 5 ml pre-hybridization solution (the same as used for hybridization but without the radioactive sample) at 65ºC. The pre-hybridization solution was then replaced with 5 ml of the same solution containing 3X10exp6 dpm/mL of radioactive sample and hybridized for 44h. Washing conditions were: 1h at 65ºC for 20 min in 2X SSC, 0.1% SDS, and twice at 65ºC for 30 min in 0.2X SSC, 0.1% SDS.
Scan protocol
Images were acquired using a FujiFilm FLA3000 Phosphorimager. After the washing step, membranes were kept humid, sealed in Saran wrap, avoiding any bubbles, and exposed to an imaging plate (BAS-MP, FujiFilm) for various times. Filters were stripped by pouring 3X150 ml boiling stripping buffer (5 mM sodium phosphate, pH 7.5, 0.1% SDS) over the membrane. The first time, the stripping buffer was immediately changed while after the second and third washes the filters were left to cool at room temperature. To ensure that radioactivity had been eliminated, the filters were either checked with a Geiger counter or re-scanned with the Phosphorimager.
