Cultures of one colony of the appropriate strain were started in 10 mL XY (YEP supplemented with 0.02% tryptophan and 0.01% adenine) + 2% glucose and grown overnight at 30˚C. The following morning, cells were subcultured into 50 or 100 mL fresh media at an OD600 of 0.1-0.15 and grown through the day until OD600 0.7-1.0 was reached. Cultures were poured in 50mL Falcon tube(s) containing 1.4 mL 37% formaldehyde, and put on a rotating wheel at room temperature for 30 min. Crosslinking was stopped by addition of 2.5mL 2.5M glycine. For galactose induction experiments, cells grown overnight in XY + 2% glucose were washed twice in 10 mL sterile ddH2O, subcultured into XY + 2% galactose at an OD600 of 0.15 and grown for 7 h at 30˚C (final OD600 0.8-1.0) For cell synchronization experiments, cells precultured overnight in XY + 2% glucose were subcultured into 200 mL of XY pH3.9 + 2% glucose (the lower pH inhibits the Bar1 protease to make the cells more sensitive to alpha-factor) to an OD600 of 0.5-0.6. A 1 mL aliquot of cells was removed and fixed for FACS analysis in 70% ethanol (asynchronous). Alpha-factor was added to a final concentration of 5 µg/mL (stock 5 mg/mL in DMSO) and growth was continued at 30˚C for 2 h (maximum -- to prevent toxicity from DMSO). For G1-arrested cells, harvest and fix cells here (also remove a 1 mL aliquot of cells and fix for FACS analysis). To enrich for S-phase cells, cells were harvested by centrifugation, washed twice in 200 mL sterile ddH2O and resuspended in 200 mL XY + 2% glucose (standard recipe, i.e. not pH’d) and returned to the 30˚C shaker. Starting at 15 min post-release, and every 15 min after that, remove a 1 mL aliquot of cells to be fixed for FACS analysis and pour out 50 mL to be fixed for ChIP. After fixation, washing and storage of ChIP samples, analyse FACS samples by Sytox Green staining and select the timepoint of release with desired DNA content for ChIP analysis.
Extracted molecule
genomic DNA
Extraction protocol
Crosslinked cells are pelleted at 3.5k RPM in a Beckman-Coulter Allegra X-15R centrifuge (SX4750 rotor). Cells are resuspended in 40mL ice cold TBS and pelleted again. This is repeated a second time. Cell pellets are resuspended in the remaining liquid (approx. 1mL) and transfered to 2 mL screw cap tubes. Cells are pelleted by centrifugation, the supernatant removed by pipetting, and the cells quick-frozen by immersion in liquid nitrogen and stored at –80˚C. Cells are thawed on ice, and resuspended in 700 µL lysis buffer. 500uL of acid-washed glass beads (Sigma) are added, and the cells are lysed using the FastPrep FP120 (BIO101/Thermo Savant) bead-beater (8x20 sec cycles at power level 4.5, with 3min breaks on ice). The tubes are pierced with a 18-gauge syringe needle to allow elution of lysate, but not of beads. The pierced tubes are suspended in 15mL collection tubes and spun for 2 min at 1000 x g. The supernatant is removed and the pellet is resuspended to a final volume of 1 mL in lysis buffer. The lysate is sonicated (Misonix S3000) with a 5x 30 sec cycles (power 1 sec on, 1 sec off at output level 6) with a 5 minute break between sonication cycles. The sonicated lysates are centrifuged, and the supernatant is used immediately for ChIP. Lysis buffer: 50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate with protease inhibitors (Complete EDTA-free Protease Inhibitors Cocktail without EDTA; Roche) and phosphatase inhibitors (Phosphatase Inhibitor Cocktail I; Sigma). Derived from: Drouin S., Robert F. Methods (in press). For the immunoprecipitation, Anti-mouse Dynabeads or Protein G Dynabeads (50uL / ChIP) are washed three times with 1 mL PBS + 0.5% BSA (passed through 0.22 µm filter), and resuspended in 1mL PBS + 0.5% BSA. After adding the antibody at the below indicated concentration, the mixture is incubated overnight at 4C in haematological mixer. Beads are washed three times with 1mL PBS + 0.5% BSA, and resuspended in 50uL lysis buffer / ChIP. 750-850 µL of lysate are added to 50uL beads, and incubated overnight at 4C in haematological mixer. Beads are then washed (using the Dynal magnet system) twice with 1mL ice cold Lysis Buffer (no protease inhibitors), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl (720uL of 5M NaCl per 10mL lysis buffer), twice with ice cold Wash Buffer, and twice with ice cold TE. Remaining liquid is removed by pipetting, and 50uL TE + 1% SDS is added. Beads are incubated overnight at 65C to reverse the crosslinks. Note for Whole Cell Extracts (WCE): WCE are included in the protocol only at the crosslink reversal step. Add 50uL TE + 1% SDS to 20uL WCE, and put overnight at 65C to reverse the crosslinks. Lysis Buffer (no protease inhibitors): 50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate Wash Buffer: 10mM Tris-HCl, pH 8.0 250mM LiCl 0.5% NP40 0.5% Na-deoxycholate 1mM EDTA. Derived from: Drouin S., Robert F. Methods (in press). The rabbit anti-phospho-histone H2A (Ser129), yeast-specific, (Upstate Cat# 07-745, lot# 30559) was used at 1.5 uL per immunoprecipitation. The mouse 9E10 anti-c-Myc antibody (Santa Cruz) was used with 3 µl per immunoprecipitation. The rabbit anti-histone H4 antibody (AV94 RyH4) was a kind gift from Alain Verreault and was used with 2 µL per immunoprecipitation. The mouse anti-RNA polymerase II CTD antibody [8WG16] (AbCam ab817) was used with 0.5 µL per immunoprecipitation. The mouse F-7 anti-HA antibody (Santa Cruz) was used with 15 µL per immunoprecipitation. To clean-up the DNA, beads are spun down and supernatant is transfered to a new 1.5mL tube. 350uL of a solution of 345uL TE / 3uL RNAse A (10mg/mL) / 2uL Glycogen (20mg/mL) is added to the supernatant and incubated at 37C for 2 hours. 15uL 10% SDS and 7.5uL 10 mg/mL Proteinase K are then added and incubated for another 2 hours at 37C. The samples are then extracted 2-3 times with 450uL phenol/chloroform/isoamyl alcohol (25:24:1) and once with chloroform/isoamyl alcohol (24:1). 16uL of 5M NaCl is added to the final extract of about 350uL. 2 volumes of freezing 100% EtOH (-20C) are added, and the samples are put at –20˚C overnight. They are then centrifuged at maximum velocity for 30 minutes at 4˚C. The supernatant is discarded, and the pellet is washed with 500uL 70% EtOH (-20C) and centrifuged 15 minutes at 4C. The supernatant is discarded, the pellet resuspended in 50uL TE and stored at -20C. Derived from: Drouin S., Robert F. Methods (in press).
Label
Cy5
Label protocol
Blunting: Do everything on ice! Add 70uL of the blunting solution (11uL NEB Buffer #2, 0.5uL BSA 10mg/mL, 0.5uL 20mM dNTPs, 0.2uL T4 DNA Polymerase 3U/uL, 57.8uL ddH2O) to 40uL of the resuspended DNA obtained at the end of the cleanup step. For WCEs, use 2uL + 38uL ddH2O instead of 40uL. Incubate 20 minutes at 12C. Add 11.5uL 3M NaOAc, pH 5.2, and 0.5uL Glycogen 20mg/mL and vortex briefly. Extract with 120uL Phenol/Chloroform/Isoamyl alcohol (25:24:1) and 120 µL Chloroform/Isoamyl alcohol (24:1). Transfer 110uL to a new tube, and add 230uL -20C 100% EtOH. Put at -20C for 60 min, then spin 30 minutes at 4C. Wash the pellet with 500uL -20C 70% EtOH and spin 15 minutes at 4C. Resuspend the pellet in 25uL ddH2O and place on ice. Ligation: Thaw everything on ice, and do all manipulations on ice in the cold room. Add 25uL of ligase mix (10uL 5x T4 Ligase Buffer, 6.7uL 15uM unidirectional linkers, 0.5uL T4 DNA Ligase, High Concentration, 8uL ddH2O) to the resuspended pellet from the blunting step. Incubate overnight at 16C. Add 6uL 3M NaOAc, pH 5.2, and vortex. Add 130uL –20˚C 100% EtOH and centrifuge 30 minutes at 4˚C. Wash the pellet (should be a smear on the side of the tube) with 500uL –20˚C 70% EtOH. Spin 15 minutes at 4C. Dry the pellet and resuspend in 25uL ddH2O. Labeling: Add 15uL of PCR Labeling mix (4uL 10x ThermoPol Buffer, 2uL 5mM aa-dUTP mix, 1.25uL Oligo FR1, 7.75uL ddH2O) to the pellet from the ligation step. Start the PCR program, and after 1 min of the first step (4 minutes at 55˚C) pause the program and add 10uL of the polymerase mix (1uL 10x ThermoPol buffer, 1uL Taq Polymerase 5U/uL, 0.01uL Pfu Polymerase 2.5U/uL, 8uL ddH2O) before continuing the program. PCR cycling conditions: 1) 4:00 at 55C 2) 5:00 at 72C 3) 2:00 at 95C 4) 0:30 at 95C 5) 0:30 at 55C 6) 1:00 at 72C 7) Repeat steps 4-6 31 more times. 8) 4:00 at 72C 9) Forever at 4C Purify PCR reactions with Qiagen PCR Cleanup kits, following their protocol BUT use a phosphate wash buffer and a phosphate elution buffer (see below) instead of their buffers PE and EB, respectively. Also, wash the columns twice (first time immediately after adding wash buffer, second time after 5 min incubation), and elute twice with 30uL elution buffer. Speed-vac the eluate to dryness and store at –20˚C if desired. Resuspend the pellets with freshly made 4.5uL 0.1M Sodium Carbonate Buffer, pH 9.0. Add 4.5uL of the appropriate NHS-ester Cy-dye and place in the dark. Fresh tubes of NHS-ester Cy-dye should be resuspended in 73uL DMSO. Mix well, and incubate at room temperature in the dark for one hour. Add 35uL 0.1M NaOAc, pH 5.2. Protecting samples from light as much as reasonably possible, purify using the Qiagen PCR Cleanup protocol using all their buffers, but wash the columns twice (first time immediately after adding wash buffer, second time after 5 min incubation), and elute twice in 30uL EB. It is a good idea to mix both samples of a pair (Cy5 and Cy3) in the same tube to avoid DNA loss upon later resuspension. Speed-vac until dryness (cover the chamber with tinfoil). Phosphate Wash Buffer (100mL): 0.5mL 1M KPO4, pH 8.5 15.25mL ddH2O 84.25mL 95% EtOH Solution will be cloudy upon mixing. Phosphate Elution Buffer (10mL): 0.04mL 1M KPO4, pH 8.5 9.96mL ddH2O 1M KPO4, pH 8.5 (10mL): 9.5mL 1M K2HPO4 0.5mL 1M KH2PO4 0.1M Sodium Carbonate Buffer, pH 9.0 (50mL): 0.53g Na2CO3 + 40mL ddH2O pH to 9.0 with concentrated HCl Adjust volume to 50mL with ddH2O and pass through 0.22 µm filter. Make fresh each time. Derived from: Drouin S., Robert F. Methods (in press).
Cultures of one colony of the appropriate strain were started in 10 mL XY (YEP supplemented with 0.02% tryptophan and 0.01% adenine) + 2% glucose and grown overnight at 30˚C. The following morning, cells were subcultured into 50 or 100 mL fresh media at an OD600 of 0.1-0.15 and grown through the day until OD600 0.7-1.0 was reached. Cultures were poured in 50mL Falcon tube(s) containing 1.4 mL 37% formaldehyde, and put on a rotating wheel at room temperature for 30 min. Crosslinking was stopped by addition of 2.5mL 2.5M glycine. For galactose induction experiments, cells grown overnight in XY + 2% glucose were washed twice in 10 mL sterile ddH2O, subcultured into XY + 2% galactose at an OD600 of 0.15 and grown for 7 h at 30˚C (final OD600 0.8-1.0) For cell synchronization experiments, cells precultured overnight in XY + 2% glucose were subcultured into 200 mL of XY pH3.9 + 2% glucose (the lower pH inhibits the Bar1 protease to make the cells more sensitive to alpha-factor) to an OD600 of 0.5-0.6. A 1 mL aliquot of cells was removed and fixed for FACS analysis in 70% ethanol (asynchronous). Alpha-factor was added to a final concentration of 5 µg/mL (stock 5 mg/mL in DMSO) and growth was continued at 30˚C for 2 h (maximum -- to prevent toxicity from DMSO). For G1-arrested cells, harvest and fix cells here (also remove a 1 mL aliquot of cells and fix for FACS analysis). To enrich for S-phase cells, cells were harvested by centrifugation, washed twice in 200 mL sterile ddH2O and resuspended in 200 mL XY + 2% glucose (standard recipe, i.e. not pH’d) and returned to the 30˚C shaker. Starting at 15 min post-release, and every 15 min after that, remove a 1 mL aliquot of cells to be fixed for FACS analysis and pour out 50 mL to be fixed for ChIP. After fixation, washing and storage of ChIP samples, analyse FACS samples by Sytox Green staining and select the timepoint of release with desired DNA content for ChIP analysis.
Extracted molecule
genomic DNA
Extraction protocol
Crosslinked cells are pelleted at 3.5k RPM in a Beckman-Coulter Allegra X-15R centrifuge (SX4750 rotor). Cells are resuspended in 40mL ice cold TBS and pelleted again. This is repeated a second time. Cell pellets are resuspended in the remaining liquid (approx. 1mL) and transfered to 2 mL screw cap tubes. Cells are pelleted by centrifugation, the supernatant removed by pipetting, and the cells quick-frozen by immersion in liquid nitrogen and stored at –80˚C. Cells are thawed on ice, and resuspended in 700 µL lysis buffer. 500uL of acid-washed glass beads (Sigma) are added, and the cells are lysed using the FastPrep FP120 (BIO101/Thermo Savant) bead-beater (8x20 sec cycles at power level 4.5, with 3min breaks on ice). The tubes are pierced with a 18-gauge syringe needle to allow elution of lysate, but not of beads. The pierced tubes are suspended in 15mL collection tubes and spun for 2 min at 1000 x g. The supernatant is removed and the pellet is resuspended to a final volume of 1 mL in lysis buffer. The lysate is sonicated (Misonix S3000) with a 5x 30 sec cycles (power 1 sec on, 1 sec off at output level 6) with a 5 minute break between sonication cycles. The sonicated lysates are centrifuged, and the supernatant is used immediately for ChIP. Lysis buffer: 50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate with protease inhibitors (Complete EDTA-free Protease Inhibitors Cocktail without EDTA; Roche) and phosphatase inhibitors (Phosphatase Inhibitor Cocktail I; Sigma). Derived from: Drouin S., Robert F. Methods (in press). For the immunoprecipitation, Anti-mouse Dynabeads or Protein G Dynabeads (50uL / ChIP) are washed three times with 1 mL PBS + 0.5% BSA (passed through 0.22 µm filter), and resuspended in 1mL PBS + 0.5% BSA. After adding the antibody at the below indicated concentration, the mixture is incubated overnight at 4C in haematological mixer. Beads are washed three times with 1mL PBS + 0.5% BSA, and resuspended in 50uL lysis buffer / ChIP. 750-850 µL of lysate are added to 50uL beads, and incubated overnight at 4C in haematological mixer. Beads are then washed (using the Dynal magnet system) twice with 1mL ice cold Lysis Buffer (no protease inhibitors), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl (720uL of 5M NaCl per 10mL lysis buffer), twice with ice cold Wash Buffer, and twice with ice cold TE. Remaining liquid is removed by pipetting, and 50uL TE + 1% SDS is added. Beads are incubated overnight at 65C to reverse the crosslinks. Note for Whole Cell Extracts (WCE): WCE are included in the protocol only at the crosslink reversal step. Add 50uL TE + 1% SDS to 20uL WCE, and put overnight at 65C to reverse the crosslinks. Lysis Buffer (no protease inhibitors): 50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate Wash Buffer: 10mM Tris-HCl, pH 8.0 250mM LiCl 0.5% NP40 0.5% Na-deoxycholate 1mM EDTA. Derived from: Drouin S., Robert F. Methods (in press). The rabbit anti-phospho-histone H2A (Ser129), yeast-specific, (Upstate Cat# 07-745, lot# 30559) was used at 1.5 uL per immunoprecipitation. The mouse 9E10 anti-c-Myc antibody (Santa Cruz) was used with 3 µl per immunoprecipitation. The rabbit anti-histone H4 antibody (AV94 RyH4) was a kind gift from Alain Verreault and was used with 2 µL per immunoprecipitation. The mouse anti-RNA polymerase II CTD antibody [8WG16] (AbCam ab817) was used with 0.5 µL per immunoprecipitation. The mouse F-7 anti-HA antibody (Santa Cruz) was used with 15 µL per immunoprecipitation. To clean-up the DNA, beads are spun down and supernatant is transfered to a new 1.5mL tube. 350uL of a solution of 345uL TE / 3uL RNAse A (10mg/mL) / 2uL Glycogen (20mg/mL) is added to the supernatant and incubated at 37C for 2 hours. 15uL 10% SDS and 7.5uL 10 mg/mL Proteinase K are then added and incubated for another 2 hours at 37C. The samples are then extracted 2-3 times with 450uL phenol/chloroform/isoamyl alcohol (25:24:1) and once with chloroform/isoamyl alcohol (24:1). 16uL of 5M NaCl is added to the final extract of about 350uL. 2 volumes of freezing 100% EtOH (-20C) are added, and the samples are put at –20˚C overnight. They are then centrifuged at maximum velocity for 30 minutes at 4˚C. The supernatant is discarded, and the pellet is washed with 500uL 70% EtOH (-20C) and centrifuged 15 minutes at 4C. The supernatant is discarded, the pellet resuspended in 50uL TE and stored at -20C. Derived from: Drouin S., Robert F. Methods (in press).
Label
Cy3
Label protocol
Blunting: Do everything on ice! Add 70uL of the blunting solution (11uL NEB Buffer #2, 0.5uL BSA 10mg/mL, 0.5uL 20mM dNTPs, 0.2uL T4 DNA Polymerase 3U/uL, 57.8uL ddH2O) to 40uL of the resuspended DNA obtained at the end of the cleanup step. For WCEs, use 2uL + 38uL ddH2O instead of 40uL. Incubate 20 minutes at 12C. Add 11.5uL 3M NaOAc, pH 5.2, and 0.5uL Glycogen 20mg/mL and vortex briefly. Extract with 120uL Phenol/Chloroform/Isoamyl alcohol (25:24:1) and 120 µL Chloroform/Isoamyl alcohol (24:1). Transfer 110uL to a new tube, and add 230uL -20C 100% EtOH. Put at -20C for 60 min, then spin 30 minutes at 4C. Wash the pellet with 500uL -20C 70% EtOH and spin 15 minutes at 4C. Resuspend the pellet in 25uL ddH2O and place on ice. Ligation: Thaw everything on ice, and do all manipulations on ice in the cold room. Add 25uL of ligase mix (10uL 5x T4 Ligase Buffer, 6.7uL 15uM unidirectional linkers, 0.5uL T4 DNA Ligase, High Concentration, 8uL ddH2O) to the resuspended pellet from the blunting step. Incubate overnight at 16C. Add 6uL 3M NaOAc, pH 5.2, and vortex. Add 130uL –20˚C 100% EtOH and centrifuge 30 minutes at 4˚C. Wash the pellet (should be a smear on the side of the tube) with 500uL –20˚C 70% EtOH. Spin 15 minutes at 4C. Dry the pellet and resuspend in 25uL ddH2O. Labeling: Add 15uL of PCR Labeling mix (4uL 10x ThermoPol Buffer, 2uL 5mM aa-dUTP mix, 1.25uL Oligo FR1, 7.75uL ddH2O) to the pellet from the ligation step. Start the PCR program, and after 1 min of the first step (4 minutes at 55˚C) pause the program and add 10uL of the polymerase mix (1uL 10x ThermoPol buffer, 1uL Taq Polymerase 5U/uL, 0.01uL Pfu Polymerase 2.5U/uL, 8uL ddH2O) before continuing the program. PCR cycling conditions: 1) 4:00 at 55C 2) 5:00 at 72C 3) 2:00 at 95C 4) 0:30 at 95C 5) 0:30 at 55C 6) 1:00 at 72C 7) Repeat steps 4-6 31 more times. 8) 4:00 at 72C 9) Forever at 4C Purify PCR reactions with Qiagen PCR Cleanup kits, following their protocol BUT use a phosphate wash buffer and a phosphate elution buffer (see below) instead of their buffers PE and EB, respectively. Also, wash the columns twice (first time immediately after adding wash buffer, second time after 5 min incubation), and elute twice with 30uL elution buffer. Speed-vac the eluate to dryness and store at –20˚C if desired. Resuspend the pellets with freshly made 4.5uL 0.1M Sodium Carbonate Buffer, pH 9.0. Add 4.5uL of the appropriate NHS-ester Cy-dye and place in the dark. Fresh tubes of NHS-ester Cy-dye should be resuspended in 73uL DMSO. Mix well, and incubate at room temperature in the dark for one hour. Add 35uL 0.1M NaOAc, pH 5.2. Protecting samples from light as much as reasonably possible, purify using the Qiagen PCR Cleanup protocol using all their buffers, but wash the columns twice (first time immediately after adding wash buffer, second time after 5 min incubation), and elute twice in 30uL EB. It is a good idea to mix both samples of a pair (Cy5 and Cy3) in the same tube to avoid DNA loss upon later resuspension. Speed-vac until dryness (cover the chamber with tinfoil). Phosphate Wash Buffer (100mL): 0.5mL 1M KPO4, pH 8.5 15.25mL ddH2O 84.25mL 95% EtOH Solution will be cloudy upon mixing. Phosphate Elution Buffer (10mL): 0.04mL 1M KPO4, pH 8.5 9.96mL ddH2O 1M KPO4, pH 8.5 (10mL): 9.5mL 1M K2HPO4 0.5mL 1M KH2PO4 0.1M Sodium Carbonate Buffer, pH 9.0 (50mL): 0.53g Na2CO3 + 40mL ddH2O pH to 9.0 with concentrated HCl Adjust volume to 50mL with ddH2O and pass through 0.22 µm filter. Make fresh each time. Derived from: Drouin S., Robert F. Methods (in press).
Hybridization protocol
Resuspend Cy5 labeled sample in 4ul of water and combine with corresponding Cy3 labeled sample. Mix to 450ul of the hybridization buffer for each slide (45ul 0.5M Mes NaOH (pH 6.9), 135ul Formamide, 58.5ul 5M NaCl, 45ul 5% N-Lauroylsarcosine (FLUKA), 1ul 250 ug/ml Salmon Sperm DNA, 10ul 8 mg/ml yeast tRNA, 5.4ul 0.5 M EDTA, 150.1ul mQH2O). Boil samples for 3 minutes in 100ºC heat block. Transfer to 40ºC heat block for another 10 minutes. Follow standard Agilent chamber hybridization protocols. Incubate overnight at 40C in hybridization oven. Post-Hybridization Washes: Separate coverslip and slide in Wash buffer #1. Wash slides in Wash buffer #1 for 5 minutes at room temperature on an orbital shaker (cover the dish to avoid light on the slides). Wash in 31C Wash buffer #2 for 5 minutes on an orbital shaker. Take slides out of the buffer slowly to dry them. Wash Buffer #1: 6x SSPE, 0.005% N-lauroylsarcosine. Wash Buffer #2: 0.06x SSPE. Stripping is done in 1 L of 5 mM Potassium phosphate buffer pH 6.6. 1. Use a 2L beaker, which can fit a slide rack. Pour the stripping buffer, submerge the rack with slides (from 1 to 10) and heat it up slowly while stirring with a stir bar until the liquid boils with big bubbles. Do not overboil it. Remove the rack and briefly rinse in a container with water. Slowly remove the rack from the liquid. Cy5 channel will be stripped much better than Cy3, but this does not affect the performance of subsequent hybridizations. Stock solution of 1M Potassium phosphate buffer, pH 6.6: Mix 3.81 ml of 1M K2HPO4 + 6.19 ml of 1M KH2PO4. Dilute 2.5 ml of this 1M solution in 500 ml Milli-Q water to obtain 5mM Potassium phosphate buffer, pH 6.6.
Scan protocol
Axon GenePix 4000B scanner and the GenePix Pro extraction software were used. Scan at 100% laser power for both channels. Set the Pixel Size to 5um / pixel. Set the Lines to average to 1. Set the Focus Position to 0um. Adjust PMTs so that approximately 1-2% spots are saturated. This is done to ensure full dynamic range utilization. Adjust PMTs so that the intensity ratio is 0.9 - 1.1 when looking at intensity values greater than or equal to 3000 on the Intensity / Frequence histogram. Images were quantified using Axon GenePix (version 6.0 and 6.1).
Description
Biological replicate 2 of 2. H2AS129pi enrichment in the suppressor of mec1 lethality sml1 delta measured in two cell lines by the ratio sml1D/sml1D_S129A.
Data processing
The raw data were corrected (foreground-background) then normalized using the limma's loess function (Yang et al.,2002) in BioConductor (from the ArrayPipe Analysis Pipeline (Hokamp et al., 2004)) and replicates were combined using a weighted average method as described previously (Pokholok et al., 2005).
