strain: yFR551 antibody: 3E8 (Ser5) vendor: Helmholtz Zentrum
Growth protocol
Culture were done at 30C in YEP+2% Glucose media (50 to 200mL) inoculated at OD600 0.1 from an overnight preculture in YEP+2% Glucose media. Culture were allowed to grow until it reach OD600 0.6-1.0, poured in 50mL Falcon tubes containing 1.4mL 37% formaldehyde for crosslinking (1% final), and incubate on rotating wheel for 30 min at room temperature. Crosslinking was stopped by addition of 2.5mL 2.5M Glycine to quench formaldehyde (~125mM final). Crosslinked cells were pelleted at 4 C, 3000 rpm ( in Beckman centrifuge (GS 6R Rotor 3.8)), resuspended in 40mL ice cold TBS and pelleted again. This wash was repeated a second time. Cell pellets were resuspended in the remaining liquid (~1mL) and transferred to 1.5mL screw cap tubes. Cells were pelleted by centrifugation, the supernatant removed by pipetting, and the cells snap-freeze in liquid nitrogen. Cells were store at -80 C. Ref: Bataille A.R., Robert F. Methods in Enzymology 2009
Extracted molecule
genomic DNA
Extraction protocol
Cells are thawed on ice, and resuspended in 700mL lysis buffer with protease inhibitors (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). 500uL of acid-washed glass beads (Sigma) are added, and the cells lysed using the bead-beater (4x5min cycles, with 5min breaks on ice). The tubes are pierced with a 18-gauge syringe needle and place in 2mL collection tubes to allow transfer of lysate, but not beads, with a brief centrifugation. The lysates are then homogenized by pipetting and sonicated 4x 20sec at output 7 Watts, with a 1 minute break between sonication cycles. The sonicated lysates are centrifuge for 5 min at 4 C max speed and supernatant used immediately for ChIP (or snap-freeze in liquid nitrogen and saved frozen at -80C). For immunoprecipitation, Dynabeads (details at the end of the section) (50uL / ChIP) are washed twice with PBS + 0.5% BSA (freshly made), and resuspended in PBS + 0.5% BSA. Antibody are added (Concentration and souce at the end of the section), and incubated overnight at 4 C in haematological mixer. Beads are washed twice (with PBS/BSA or Lysis buffer), and resuspended in 30uL (PBS/BSA or Lysis buffer) / ChIP. 30uL of resuspended beads/antibody are added to 400-600uL of lysate, and incubated overnight at 4 C in haematological mixer. Beads are then washed in cold room (using the Dynal magnet system) twice with 1mL ice cold Lysis Buffer (no protease inhibitors)(50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate ), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer (10mM Tris-HCl, pH 8.0 250mM LiCl 0.5% NP40 0.5% Na-deoxycholate 1mM EDTA), and once with ice cold TE. Remaining liquid is removed by pipetting after 1 min centrifugation at 4 C (max speed). 50uL TE + 1% SDS is added and beads are incubated overnight at 65 C for elution and reverse crosslink. Note for Whole Cell Extracts (WCE): WCE are included in the protocol only at the crosslink reversal step. Add 50uL TE + 1% SDS to 5uL WCE, and put overnight at 65C to reverse the crosslinks with the ChIP samples. For this study the anti-myc (9E10) was a kind gift from Alain Verreault. The anti-Flag (M2) was from Sigma. The 3E8, 3E10 and 4E12 antibodies were purchased from Helmholtz Zentrum in Munich. The H5 (P-Ser2) and H14 (P-Ser5) were purchased from Covance. The anti-Rpb3 (W0012) was purchased from Neoclone Biotechnology. The 8WG16 (Ab817) antibody was purchased from Abcam.The following amounts of antibody per immunoprecipitation were used: 3E8 (5μL), 3E10 (5μL), 4E12 (5μL), 8WG16 (2μg), H14 (15μg), H5 (15μg), W0012 (3μL), 9E10 (5μg), M2 (5μg). The antibodies were coupled to Dynabeads (Invitrogen) coated with protein G (3E8, 3E10, 4E12), IgG Pan mouse antibodies (8WG16, H14, W0012, 9E10, M2) or Sheep anti-mouse IgG (H5). 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 samples incubated at 37C for 2 hours. 15uL of 10% SDS and 7.5uL Proteinase K are then added and the tubes incubated for another 2 hours at 37 C. The samples are then extracted twice with 400uL phenol/chloroform/isoamyl alcohol (25:24:1) and precipitated with 14uL of 5M NaCl (for about 350uL of extracted sample) and 1mL of freezing 100% EtOH (-20 C). Pellets are washed with 700uL 70% EtOH (-20 C) and left to air dry for few minutes before they are resuspended in 50uL TE. Stored ChIP samples at -20 C. Ref: Bataille A.R., Robert F. Methods in Enzymology 2009.
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 mQH2O) to 40uL of the resuspended DNA obtained at the end of the cleanup step. For WCEs, use 400ng of DNA diluted to 40uL with mQH2O. Incubate 20 minutes at 12C. Add 12uL of a the following mix: 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 -20'C for at least 30 min, then spin 20 minutes at 4C. Wash the pellet with 700uL -20'C 70% EtOH and spin 15 minutes at 4C. Resuspend the pellet in 25uL mQH2O and place on ice. Ligation: Thaw everything on ice, and do all manipulations on ice (or in 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 mQH2O) 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 20 minutes at 4C. Wash the pellet (visible as a smear on the side of the tube) with 700uL -20C 70% EtOH. Spin 15 minutes at 4C. Dry the pellet and resuspend in 25uL mQH2O. Labeling: Add 15uL of PCR aa-dUTP mix (4uL 10x ThermoPol Buffer, 2uL 5mM aa-dUTP mix, 1.25uL Oligo FR1, 7.75uL mQH2O) to resuspended ligation samples. 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 mQH2O)(Pause program if necessary). 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 elute twice with 40uL 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 (Use DMSO store in vacuum with desiccant). Resuspended dyes are store in the dark at -80?C. 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, elute twice in 40uL. Speed-vac until ~10uL is left, or to dryness (corresponding Cy3 and Cy5 samples can be mixed before speed-vac in order to avoid DNA loss upon later resuspension). Ref: Bataille A.R., Robert F. Methods in Enzymology 2009.
Culture were done at 30C in YEP+2% Glucose media (50 to 200mL) inoculated at OD600 0.1 from an overnight preculture in YEP+2% Glucose media. Culture were allowed to grow until it reach OD600 0.6-1.0, poured in 50mL Falcon tubes containing 1.4mL 37% formaldehyde for crosslinking (1% final), and incubate on rotating wheel for 30 min at room temperature. Crosslinking was stopped by addition of 2.5mL 2.5M Glycine to quench formaldehyde (~125mM final). Crosslinked cells were pelleted at 4 C, 3000 rpm ( in Beckman centrifuge (GS 6R Rotor 3.8)), resuspended in 40mL ice cold TBS and pelleted again. This wash was repeated a second time. Cell pellets were resuspended in the remaining liquid (~1mL) and transferred to 1.5mL screw cap tubes. Cells were pelleted by centrifugation, the supernatant removed by pipetting, and the cells snap-freeze in liquid nitrogen. Cells were store at -80 C. Ref: Bataille A.R., Robert F. Methods in Enzymology 2009
Extracted molecule
genomic DNA
Extraction protocol
Cells are thawed on ice, and resuspended in 700mL lysis buffer with protease inhibitors (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). 500uL of acid-washed glass beads (Sigma) are added, and the cells lysed using the bead-beater (4x5min cycles, with 5min breaks on ice). The tubes are pierced with a 18-gauge syringe needle and place in 2mL collection tubes to allow transfer of lysate, but not beads, with a brief centrifugation. The lysates are then homogenized by pipetting and sonicated 4x 20sec at output 7 Watts, with a 1 minute break between sonication cycles. The sonicated lysates are centrifuge for 5 min at 4 C max speed and supernatant used immediately for ChIP (or snap-freeze in liquid nitrogen and saved frozen at -80C). For immunoprecipitation, Dynabeads (details at the end of the section) (50uL / ChIP) are washed twice with PBS + 0.5% BSA (freshly made), and resuspended in PBS + 0.5% BSA. Antibody are added (Concentration and souce at the end of the section), and incubated overnight at 4 C in haematological mixer. Beads are washed twice (with PBS/BSA or Lysis buffer), and resuspended in 30uL (PBS/BSA or Lysis buffer) / ChIP. 30uL of resuspended beads/antibody are added to 400-600uL of lysate, and incubated overnight at 4 C in haematological mixer. Beads are then washed in cold room (using the Dynal magnet system) twice with 1mL ice cold Lysis Buffer (no protease inhibitors)(50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate ), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer (10mM Tris-HCl, pH 8.0 250mM LiCl 0.5% NP40 0.5% Na-deoxycholate 1mM EDTA), and once with ice cold TE. Remaining liquid is removed by pipetting after 1 min centrifugation at 4 C (max speed). 50uL TE + 1% SDS is added and beads are incubated overnight at 65 C for elution and reverse crosslink. Note for Whole Cell Extracts (WCE): WCE are included in the protocol only at the crosslink reversal step. Add 50uL TE + 1% SDS to 5uL WCE, and put overnight at 65C to reverse the crosslinks with the ChIP samples. For this study the anti-myc (9E10) was a kind gift from Alain Verreault. The anti-Flag (M2) was from Sigma. The 3E8, 3E10 and 4E12 antibodies were purchased from Helmholtz Zentrum in Munich. The H5 (P-Ser2) and H14 (P-Ser5) were purchased from Covance. The anti-Rpb3 (W0012) was purchased from Neoclone Biotechnology. The 8WG16 (Ab817) antibody was purchased from Abcam.The following amounts of antibody per immunoprecipitation were used: 3E8 (5μL), 3E10 (5μL), 4E12 (5μL), 8WG16 (2μg), H14 (15μg), H5 (15μg), W0012 (3μL), 9E10 (5μg), M2 (5μg). The antibodies were coupled to Dynabeads (Invitrogen) coated with protein G (3E8, 3E10, 4E12), IgG Pan mouse antibodies (8WG16, H14, W0012, 9E10, M2) or Sheep anti-mouse IgG (H5). 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 samples incubated at 37C for 2 hours. 15uL of 10% SDS and 7.5uL Proteinase K are then added and the tubes incubated for another 2 hours at 37 C. The samples are then extracted twice with 400uL phenol/chloroform/isoamyl alcohol (25:24:1) and precipitated with 14uL of 5M NaCl (for about 350uL of extracted sample) and 1mL of freezing 100% EtOH (-20 C). Pellets are washed with 700uL 70% EtOH (-20 C) and left to air dry for few minutes before they are resuspended in 50uL TE. Stored ChIP samples at -20 C. Ref: Bataille A.R., Robert F. Methods in Enzymology 2009.
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 mQH2O) to 40uL of the resuspended DNA obtained at the end of the cleanup step. For WCEs, use 400ng of DNA diluted to 40uL with mQH2O. Incubate 20 minutes at 12C. Add 12uL of a the following mix: 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 -20'C for at least 30 min, then spin 20 minutes at 4C. Wash the pellet with 700uL -20'C 70% EtOH and spin 15 minutes at 4C. Resuspend the pellet in 25uL mQH2O and place on ice. Ligation: Thaw everything on ice, and do all manipulations on ice (or in 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 mQH2O) 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 20 minutes at 4C. Wash the pellet (visible as a smear on the side of the tube) with 700uL -20C 70% EtOH. Spin 15 minutes at 4C. Dry the pellet and resuspend in 25uL mQH2O. Labeling: Add 15uL of PCR aa-dUTP mix (4uL 10x ThermoPol Buffer, 2uL 5mM aa-dUTP mix, 1.25uL Oligo FR1, 7.75uL mQH2O) to resuspended ligation samples. 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 mQH2O)(Pause program if necessary). 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 elute twice with 40uL 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 (Use DMSO store in vacuum with desiccant). Resuspended dyes are store in the dark at -80?C. 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, elute twice in 40uL. Speed-vac until ~10uL is left, or to dryness (corresponding Cy3 and Cy5 samples can be mixed before speed-vac in order to avoid DNA loss upon later resuspension). Ref: Bataille A.R., Robert F. Methods in Enzymology 2009.
Hybridization protocol
Hybridization: Add 110uL of hybridization buffer (100uL DIGEasy Buffer, 5uL 10mg/mL Salmon Sperm DNA, 5uL 8mg/mL yeast tRNA) to the resuspended (mixed) colored pellet. Put at 95C for 5 minutes, centrifuge briefly and transfer at 42C for 10 min (Keep samples at 42C while waiting for hybridization). Follow standard Agilent chamber hybridization protocols. Incubate overnight (16-24h) at 42C 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. Ref: Bataille A.R., Robert F. Methods in Enzymology 2009. 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). Submerge the rack with slides (from 1 to 10) in stripping buffer 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.
Scan protocol
Scanner Axon GenePix4000B and GenePix Pro (version 6.1.0.4) extraction software were used for scanning and features quantification. 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. Set the minimal intensity (histogram preview) to 3000-5000. Adjust PMTs so that approximately 5-10% 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 possible).
Description
Biological replicate 2 of 2. RNAPII CTD P-Ser5 occupancy, using 3E8 antibody, measured by the IP/input ratio in yFR551 ctk1D 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).
