For chromatin immunoprecipitation, saturated overnight cultures were subcultured in 100 ml of synthetic complete (SC) media lacking isoleucine/valine and were grown to A600 between 0.5 and 0.6. The cultures were treated with 0.65 μg/ml of sulfometuron methyl (SM) for 25 minutes to induce Gcn4. Analog sensitive Ser2 kinase mutants (bur1as/ctk1Δ) were treated with 6 μM of 3MB-PP1 one hour prior to addition of SM as described previously (Qiu et al., 2009).
Extracted molecule
genomic DNA
Extraction protocol
Chromatin immunoprecipitation experiments were performed as described previously (Govind et al., 2012). Briefly, 100 ml of cells (A600 = 0.6) were cross-linked with 1% formaldehyde for 15 minutes at ambient temperature and quenched with glycine. Chromatin was isolated and fragmented by sonication (Branson450) to an average size of 300-400 base pairs. The soluble fraction of chromatin was used for ChIP using the appropriate antibodies.
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). Transfer 110uL to a new tube, and add 230uL -20C 100% EtOH. Put at -80C for 30min, then spin 20 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. 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 -20C 100% EtOH and centrifuge 20 minutes at 4C. Wash the pellet (should be a smear on the side of the tube) with 500uL -20C 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 during the first step (4 minutes at 55C), add 10uL of the polymerase mix (1uL 10x ThermoPol buffer, 1uL Taq Polymerase 5U/uL, 0.01uL Pfu Polymerase Turbo 2.5U/uL, 8uL ddH2O). PCR cycling conditions: 1) 5:00 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 instead of their buffers PE and EB, respectively. Also, wash the columns only once, and elute twice with 30uL elution buffer. Speed-vac the eluate to dryness. Resuspend the pellets with fresh 4.5uL 0.1M Sodium Carbonate Buffer, pH 9.0. Add 4.5uL of the appropriate NHS-ester Cy-dye. 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. Purify using the Qiagen PCR Cleanup protocol using all their buffers, but wash only once and elute twice in 30uL. It is a good idea to elute both samples of a pair (Cy5 and Cy3) in the same tube to avoid DNA loss upon later resuspension. Speed-vac until 10-20uL are left, or to dryness. 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 1M Sodium Carbonate Buffer, pH 9.0 (100mL): 10.8g Na2CO3 80mL ddH2O pH to 9.0 with concentrated HCl Adjust volume to 100mL with ddH2O DILUTE 1:10 WHEN USING! Solution will change composition over time. Use only if less than a month old.
For chromatin immunoprecipitation, saturated overnight cultures were subcultured in 100 ml of synthetic complete (SC) media lacking isoleucine/valine and were grown to A600 between 0.5 and 0.6. The cultures were treated with 0.65 μg/ml of sulfometuron methyl (SM) for 25 minutes to induce Gcn4. Analog sensitive Ser2 kinase mutants (bur1as/ctk1Δ) were treated with 6 μM of 3MB-PP1 one hour prior to addition of SM as described previously (Qiu et al., 2009).
Extracted molecule
genomic DNA
Extraction protocol
Chromatin immunoprecipitation experiments were performed as described previously (Govind et al., 2012). Briefly, 100 ml of cells (A600 = 0.6) were cross-linked with 1% formaldehyde for 15 minutes at ambient temperature and quenched with glycine. Chromatin was isolated and fragmented by sonication (Branson450) to an average size of 300-400 base pairs. The soluble fraction of chromatin was used for ChIP using the appropriate antibodies.
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). Transfer 110uL to a new tube, and add 230uL -20C 100% EtOH. Put at -80C for 30min, then spin 20 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. 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 -20C 100% EtOH and centrifuge 20 minutes at 4C. Wash the pellet (should be a smear on the side of the tube) with 500uL -20C 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 during the first step (4 minutes at 55C), add 10uL of the polymerase mix (1uL 10x ThermoPol buffer, 1uL Taq Polymerase 5U/uL, 0.01uL Pfu Polymerase Turbo 2.5U/uL, 8uL ddH2O). PCR cycling conditions: 1) 5:00 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 instead of their buffers PE and EB, respectively. Also, wash the columns only once, and elute twice with 30uL elution buffer. Speed-vac the eluate to dryness. Resuspend the pellets with fresh 4.5uL 0.1M Sodium Carbonate Buffer, pH 9.0. Add 4.5uL of the appropriate NHS-ester Cy-dye. 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. Purify using the Qiagen PCR Cleanup protocol using all their buffers, but wash only once and elute twice in 30uL. It is a good idea to elute both samples of a pair (Cy5 and Cy3) in the same tube to avoid DNA loss upon later resuspension. Speed-vac until 10-20uL are left, or to dryness. 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 1M Sodium Carbonate Buffer, pH 9.0 (100mL): 10.8g Na2CO3 80mL ddH2O pH to 9.0 with concentrated HCl Adjust volume to 100mL with ddH2O DILUTE 1:10 WHEN USING! Solution will change composition over time. Use only if less than a month old.
Hybridization protocol
Hybridization: Resuspend colored pellet in 110uL hybridization buffer (100uL DIGEasy Buffer, 5uL 10mg/mL Salmon Sperm DNA, 5uL 8mg/mL yeast tRNA). Put at 95?C for 5 minutes. Put at 42?C for 10 min (Keep samples at 42?C while waiting for hybridization). Follow standard Agilent chamber hybridization protocols. Incubate overnight (16-24h) at 42?C in Agilent hybridization oven at 20 rpm rotation speed. 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: Stripping is done in 1L of 5 mM Potassium phosphate buffer pH 6.6 after scanning of the slides. 1. Use a crystallization dish, and a metallic slide rack (use weighing spatulas to lift the rack from the bottom of the dish and avoid direct contact with the hot surface). Pour the stripping buffer, submerge the rack with slides (from 1 to 10) and heat it up slowly on heating plate until the liquid boils with big bubbles. It usually takes 15 to 20 minutes. Do not overboil it. 2. 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 5 ml of this 1M solution in 1L Milli-Q water to obtain 5mM Potassium phosphate buffer, pH 6.6.
Scan protocol
Standard_Innopsys_Yeast4x180K Set the scan as follow: pixel size = 2µm; speed = 20; Scan Mode = Normal; Acquisition Mode = Simultaneous; Focus = Auto. Adjust manually the detection gain for 635 and 532 wavelengths in order to have less than 5% spot saturation.
Description
Biological replicate 2 of 2. Spt6-MYC tagged occupancy, using the anti-MYC antibody, measured by the ratio of ChIP in tagged bur1as-ctk1D strain versus the ChIP in the untagged strain, after 60 min with 6uM 3MB-PP1.
