Cultures were started in 50mL YEP+2% Glucose from one colony of appropriate strain and incubated overnight to OD600 0.8-1.0. The NA-PP1 ATP analog was added at a final concentration of 6uM for 15min before harvesting. Cultures were poured in 50mL Falcon tube containing 1.6mL 32% paraformaldehyde, put on rotating wheel at room temperature for 30min, and then at 4C for 90min, still on the wheel. Crosslinking was stopped by addition of 2.5mL 2.5M Glycine.
Extracted molecule
genomic DNA
Extraction protocol
Crosslinked cells were washed twice with 50mL ice cold TBS, resuspended in 500uL lysis buffer (50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate 1mM PMSF 1mM Benzamidine 10ug/mL Aprotinin 1ug/mL Leupeptin 1ug/mL Pepstatin) and acid-washed glass beads were added to the meniscus. Cells were lysed using bead-beater (3x1 min, with 5min breaks on ice). The whole cell extract was mixed with 500ul of lysis buffer and centrifuged at 14000rpm for 1 minute. The pellet was resuspended in 1 ml of lysis buffer and washed twice more. The resulting extract was sonicated 6x9 minutes (30 sec on/1 min off) using a Diagenode sonicator and subsequently centrifuged at 14000rpm for 5 minutes. The supernatant containing the sonicated chromatin was transferred to a new tube. For the immunoprecipitation, beads are washed twice with 10mL PBS + 5% BSA (freshly made), and resuspended in 2mL PBS + 5% BSA. Antibodies (at the concentration indicated below) are added and incubated overnight at 4C in haematological mixer. Beads are washed twice with 10mL PBS + 5% BSA (freshly made), and resuspended in 30uL PBS + 5% BSA / ChIP. 400-500uL of lysate are added to 30uL 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 once 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 45uL TE + 1% SDS to 5uL 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. Reference: Drouin S., Robert F. Methods (in press). The 9E11 antibody (provided by Alain Verreault, IRIC) was used (5uL / ChIP) with anti-mouse Dynabeads (50uL / ChIP). To clean up the DNA, the chromatin-containing supernatant was mixed with 350ul of TE containing 3ul of RNAseA (10mg/mL) and 2ul of glycogen (20mg/mL) and incubated for 2 hours at 37C. To digest the proteins, 15ul of 10% SDS and 7.5ul Proteinase K (20mg/ml) were added and the mixture was incubated for an additional 2 hours at 37C. The samples were extracted 2x with 400uL phenol/chloroform/isoamyl alcohol (25:24:1). The DNA was precipitated with 1 mL of 100% ethanol and 14uL of 5M NaCl, the samples were centrifuged at 14,000rpm for 20 minutes at 4C. The DNA pellet was washed in 1 mL of 70% ethanol, dried and resuspended in 60ul of TE.
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 drynes+B33s. 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).
Cultures were started in 50mL YEP+2% Glucose from one colony of appropriate strain and incubated overnight to OD600 0.8-1.0. The NA-PP1 ATP analog was added at a final concentration of 6uM for 15min before harvesting. Cultures were poured in 50mL Falcon tube containing 1.6mL 32% paraformaldehyde, put on rotating wheel at room temperature for 30min, and then at 4C for 90min, still on the wheel. Crosslinking was stopped by addition of 2.5mL 2.5M Glycine.
Extracted molecule
genomic DNA
Extraction protocol
Crosslinked cells were washed twice with 50mL ice cold TBS, resuspended in 500uL lysis buffer (50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate 1mM PMSF 1mM Benzamidine 10ug/mL Aprotinin 1ug/mL Leupeptin 1ug/mL Pepstatin) and acid-washed glass beads were added to the meniscus. Cells were lysed using bead-beater (3x1 min, with 5min breaks on ice). The whole cell extract was mixed with 500ul of lysis buffer and centrifuged at 14000rpm for 1 minute. The pellet was resuspended in 1 ml of lysis buffer and washed twice more. The resulting extract was sonicated 6x9 minutes (30 sec on/1 min off) using a Diagenode sonicator and subsequently centrifuged at 14000rpm for 5 minutes. The supernatant containing the sonicated chromatin was transferred to a new tube. For the immunoprecipitation, beads are washed twice with 10mL PBS + 5% BSA (freshly made), and resuspended in 2mL PBS + 5% BSA. Antibodies (at the concentration indicated below) are added and incubated overnight at 4C in haematological mixer. Beads are washed twice with 10mL PBS + 5% BSA (freshly made), and resuspended in 30uL PBS + 5% BSA / ChIP. 400-500uL of lysate are added to 30uL 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 once 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 45uL TE + 1% SDS to 5uL 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. Reference: Drouin S., Robert F. Methods (in press). The 9E11 antibody (provided by Alain Verreault, IRIC) was used (5uL / ChIP) with anti-mouse Dynabeads (50uL / ChIP). To clean up the DNA, the chromatin-containing supernatant was mixed with 350ul of TE containing 3ul of RNAseA (10mg/mL) and 2ul of glycogen (20mg/mL) and incubated for 2 hours at 37C. To digest the proteins, 15ul of 10% SDS and 7.5ul Proteinase K (20mg/ml) were added and the mixture was incubated for an additional 2 hours at 37C. The samples were extracted 2x with 400uL phenol/chloroform/isoamyl alcohol (25:24:1). The DNA was precipitated with 1 mL of 100% ethanol and 14uL of 5M NaCl, the samples were centrifuged at 14,000rpm for 20 minutes at 4C. The DNA pellet was washed in 1 mL of 70% ethanol, dried and resuspended in 60ul of TE.
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 drynes+B33s. 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).
Hybridization protocol
Resuspend colored pellet in 110uL hybridization buffer (100uL DIGEasy Buffer, 5uL 5mg/mL Salmon Sperm DNA, 20uL 8mg/mL yeast tRNA). Put at 95C for 3 minutes. Put at 65C while waiting for hybridization. Follow standard Agilent chamber hybridization protocols. Incubate 16-20h at 40C in hybridization oven at 20RPM 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, and allow them to air dry for 1 minute. Wash Buffer #1: 6x SSPE, 0.005% N-lauroylsarcosine. Wash Buffer #2: 0.06x SSPE. Stripping is done in 500 ml 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. 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 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 2 of 2. RCO1-9Myc occupancy measured by the IP/NoTag ratio in kin28-AS 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). The combined data were next transformed to Z-scores (discarding the mitochondrial values) for the analyses.
Bioconductor packages: Biobase version 1.12.1 and limma version 2.9.1; R version 2.4.0.
