strain: yEN377 antibody: 8WG16 anti-CTD of Rpb1 (monoclonal antibody prepared in house)
Growth protocol
Cells containing TAP-tagged SEN1 or NRD1 (Ghaemmaghami et al, 2003) were grown in 50mL YPD overnight to OD600 0.8-1.0 by inoculation of a single colony. For downregulation of Sen1, the Sen1tetO7 (MATa his3Δ leu2Δ met15Δ ura3Δ URA3::CMV-tTA kanR TetO7-SEN1) (Mnaimneh et al., 2004) and its corresponding wt strains were grown in YPD containing 10ug.ml doxycycline. A volume of 5 ml of 11% formaldehyde in 0.1M NaCl, 1mM EDTA, 50 mM Hepes-KOH, pH 7.5 was added to the cultures and shaken on a rotating wheel at room temperature for 20min. Crosslinking was stopped by addition of 9 mL 3M Glycine in 20mM Tris base and incubation for 5min at room temperature.
Extracted molecule
genomic DNA
Extraction protocol
Crosslinked cells were pelleted at 3.7k RPM in Beckman centrifuge (GS 6R Rotor 3.8), then washed twice with 40mL ice cold TBS and once with 10ml ChiP lysis buffer with 1Complete tablet (50mM Hepes-KOH, pH 7.5, 150mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% sodium Deoxycholate, 0.1% SDS) before being quick-frozen by immersion in liquid nitrogen. Cells were thawed on ice, and resuspended in 1mL lysis buffer adjusted to 0.5% SDS with protease inhibitors and transferred to 1.5 ml screw cap tubes. 500uL of acid-washed glass beads (Sigma) were added, and the cells were lysed using the bead-beater (6x2.5min cycles, with 5min 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 were put in 2mL collection tubes and spun briefly to 6000 RPM. The lysate was then clarified by centrifugation for 15min at max speed in a refrigerated microfuge. The pellet was then washed with 1ml of lysis buffer containing 0.1% SDS. The pellet is then resuspended in 1ml of lysis buffer containing 0.1% SDS and the homogenate was sonicated 8x 30sec (in a ice-water bath) with a 1 minute break between sonication cycles. The sonicated lysates were centrifuged in a refrigerated centrifuge as before. After adjusting salt concentration to 275mM, the supernatant was frozen at -80C, or used immediately for ChIP. For the immunoprecipitation, 500ul of chromatin was incubated with constant agitation at 4oC overnight with either 10ul of IgG-sepharose beads prewashed with TE, for the immunoprecipitation of TAP tagged proteins or, with 1.25ul of 8WG16 monoclonal antibody prepared in house, for RNAPII immunoprecipitation. In the latter case, 20ul protein A sepharose beads were added next day and incubated for 2 hours for the collection of the immune complexes. In each case, beads containing chromatin were washed once for 4 min with rotation with 1.4 ml of the following solutions: FA lysis buffer /0.1% SDS/ 275mM NaCl (250ul 5M NaCl in 10ml). FA lysis buffer /0.1% SDS/ 500mM NaCl (700ul 5M NaCl in 10ml). 10mM Tris-HCl, pH 8.0, 0.25M LiCl, 1mM EDTA, 0.5% NP-40, 0.5% Na Deoxycholate. TE (10mM Tris-HCl, pH 8.0, 1mM EDTA). The bound proteins were eluted by incubating the beads at 65ºC for 20 minutes in 100ul of TE-SDS1%. Beads were washed with 100ul and the two eluates were combined. At this step, 20 ul of input chromatin was combined with 180uL TE + 1% SDS and treated similarly with the immunoprecipiated chromatin fraction. Chromatin fractions are deproteinized by incubation with 10ul of 10mg/ml of Proteinase K for 2hr at 37oC, followed by 6 hr at 65oC for the reversal of the crosslinks. To clean-up the DNA, Uncrosslinked chromatin fractions were then cleaned up using Qiagen QIAquick PCR Purification Kit according to the manufacturer’s instructions.
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. Reference: Drouin S., Robert F. Methods (in press).B38
Cells containing TAP-tagged SEN1 or NRD1 (Ghaemmaghami et al, 2003) were grown in 50mL YPD overnight to OD600 0.8-1.0 by inoculation of a single colony. For downregulation of Sen1, the Sen1tetO7 (MATa his3Δ leu2Δ met15Δ ura3Δ URA3::CMV-tTA kanR TetO7-SEN1) (Mnaimneh et al., 2004) and its corresponding wt strains were grown in YPD containing 10ug.ml doxycycline. A volume of 5 ml of 11% formaldehyde in 0.1M NaCl, 1mM EDTA, 50 mM Hepes-KOH, pH 7.5 was added to the cultures and shaken on a rotating wheel at room temperature for 20min. Crosslinking was stopped by addition of 9 mL 3M Glycine in 20mM Tris base and incubation for 5min at room temperature.
Extracted molecule
genomic DNA
Extraction protocol
Crosslinked cells were pelleted at 3.7k RPM in Beckman centrifuge (GS 6R Rotor 3.8), then washed twice with 40mL ice cold TBS and once with 10ml ChiP lysis buffer with 1Complete tablet (50mM Hepes-KOH, pH 7.5, 150mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% sodium Deoxycholate, 0.1% SDS) before being quick-frozen by immersion in liquid nitrogen. Cells were thawed on ice, and resuspended in 1mL lysis buffer adjusted to 0.5% SDS with protease inhibitors and transferred to 1.5 ml screw cap tubes. 500uL of acid-washed glass beads (Sigma) were added, and the cells were lysed using the bead-beater (6x2.5min cycles, with 5min 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 were put in 2mL collection tubes and spun briefly to 6000 RPM. The lysate was then clarified by centrifugation for 15min at max speed in a refrigerated microfuge. The pellet was then washed with 1ml of lysis buffer containing 0.1% SDS. The pellet is then resuspended in 1ml of lysis buffer containing 0.1% SDS and the homogenate was sonicated 8x 30sec (in a ice-water bath) with a 1 minute break between sonication cycles. The sonicated lysates were centrifuged in a refrigerated centrifuge as before. After adjusting salt concentration to 275mM, the supernatant was frozen at -80C, or used immediately for ChIP. For the immunoprecipitation, 500ul of chromatin was incubated with constant agitation at 4oC overnight with either 10ul of IgG-sepharose beads prewashed with TE, for the immunoprecipitation of TAP tagged proteins or, with 1.25ul of 8WG16 monoclonal antibody prepared in house, for RNAPII immunoprecipitation. In the latter case, 20ul protein A sepharose beads were added next day and incubated for 2 hours for the collection of the immune complexes. In each case, beads containing chromatin were washed once for 4 min with rotation with 1.4 ml of the following solutions: FA lysis buffer /0.1% SDS/ 275mM NaCl (250ul 5M NaCl in 10ml). FA lysis buffer /0.1% SDS/ 500mM NaCl (700ul 5M NaCl in 10ml). 10mM Tris-HCl, pH 8.0, 0.25M LiCl, 1mM EDTA, 0.5% NP-40, 0.5% Na Deoxycholate. TE (10mM Tris-HCl, pH 8.0, 1mM EDTA). The bound proteins were eluted by incubating the beads at 65ºC for 20 minutes in 100ul of TE-SDS1%. Beads were washed with 100ul and the two eluates were combined. At this step, 20 ul of input chromatin was combined with 180uL TE + 1% SDS and treated similarly with the immunoprecipiated chromatin fraction. Chromatin fractions are deproteinized by incubation with 10ul of 10mg/ml of Proteinase K for 2hr at 37oC, followed by 6 hr at 65oC for the reversal of the crosslinks. To clean-up the DNA, Uncrosslinked chromatin fractions were then cleaned up using Qiagen QIAquick PCR Purification Kit according to the manufacturer’s instructions.
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. Reference: Drouin S., Robert F. Methods (in press).B38
Hybridization protocol
Resuspend Cy5 labeled IP 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 6.1.0.4).
Description
Biological replicate 1 of 2. RNAPII occupancy measured by the IP/Input ratio in tetO7:sen1 cells.
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).
