20 to 40 mL of YEP+2% Glucose were inoculated with 0.4 ml of over-night culture and incubated at 30ºC to OD600 0.8-1.0. The cells were then washed twice with water, transferred in 140 ml of YEP+2% Raffinose and incubated 10 to 12 hours until they reached OD600 of 0.5. To arrest the cells in G1, alpha-Factor was added at a final concentration of 500ng/ml and the cells were incubated for 2 to 3 hours at 30ºC. For the induction of new histone H3, Galactose 2% was added directly into the media for 1 hour. The cells were crosslinked at the indicated time by adding the formaldehyde to a final concentration of 1 %.
Extracted molecule
genomic DNA
Extraction protocol
Crosslinked cells are washed twice with 50mL ice cold TBS and quickly frozen by immersion in liquid nitrogen. Subsequently, they are thawed on ice, resuspended in 250uL breaking buffer and 500uL of acid-washed glass beads are added to the mixture. Then, cells are lysed using bead-beater (2x30 sec, with 5min breaks on ice). The whole cell extract is mixed with 500ul of FA-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 centrifuged at 14000rpm for 1 minute. Then, the pellet is resuspended in 1 ml of FA-lysis buffer. This step is repeated. The resulting extract is sonicated (10X30sec) using Diagenode sonicator and subsequently centrifuged at 14000rpm for 30 minutes. The supernatant containing the sonicated chromatin is transferred to a new tube. For the immunoprecipitation, 100ul of chromatin were added to 900 ul of FA-lysis buffer. After adding the antibody at the below indicated concentration, the mixture is incubated overnight at 4 ºC on wheel. Subsequently 12.5 ul of protein G sepharose beads are added and the incubation is continued for 4-6 hours. The beads are then washed twice with 1mL ice cold FA-lysis Buffer, twice with 1mL ice cold FA-lysis Buffer (500mM NaCl), twice with ice cold ChIP 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. The bound proteins are eluted by incubating the beads at 65ºC for 20 minutes in 100ul of TE-SDS1%. To reverse the crosslinks the eluted material is incubated overnight at 65ºC. To clean-up the DNA, the protein are digested by adding 100ul of TE, 1ul of glycogen (20mg/ml) and 7.5ul Proteinase K (20mg/ml) and incubated for 1 hour at 65ºC. The samples are then extracted with 350uL phenol/chloroform/isoamyl alcohol (25:24:1). The DNA is then precipitated with 1 ml of 100% ethanol and 12.5uL of 5M NaCl. After an overnight incubated at -20ºC, the samples are centrifuged at maximum velocity for 1 hour at 4ºC. The supernatant is discarded and the pellet is resuspended in 100ul of water.
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).
20 to 40 mL of YEP+2% Glucose were inoculated with 0.4 ml of over-night culture and incubated at 30ºC to OD600 0.8-1.0. The cells were then washed twice with water, transferred in 140 ml of YEP+2% Raffinose and incubated 10 to 12 hours until they reached OD600 of 0.5. To arrest the cells in G1, alpha-Factor was added at a final concentration of 500ng/ml and the cells were incubated for 2 to 3 hours at 30ºC. For the induction of new histone H3, Galactose 2% was added directly into the media for 1 hour. The cells were crosslinked at the indicated time by adding the formaldehyde to a final concentration of 1 %.
Extracted molecule
genomic DNA
Extraction protocol
Crosslinked cells are washed twice with 50mL ice cold TBS and quickly frozen by immersion in liquid nitrogen. Subsequently, they are thawed on ice, resuspended in 250uL breaking buffer and 500uL of acid-washed glass beads are added to the mixture. Then, cells are lysed using bead-beater (2x30 sec, with 5min breaks on ice). The whole cell extract is mixed with 500ul of FA-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 centrifuged at 14000rpm for 1 minute. Then, the pellet is resuspended in 1 ml of FA-lysis buffer. This step is repeated. The resulting extract is sonicated (10X30sec) using Diagenode sonicator and subsequently centrifuged at 14000rpm for 30 minutes. The supernatant containing the sonicated chromatin is transferred to a new tube. For the immunoprecipitation, 100ul of chromatin were added to 900 ul of FA-lysis buffer. After adding the antibody at the below indicated concentration, the mixture is incubated overnight at 4 ºC on wheel. Subsequently 12.5 ul of protein G sepharose beads are added and the incubation is continued for 4-6 hours. The beads are then washed twice with 1mL ice cold FA-lysis Buffer, twice with 1mL ice cold FA-lysis Buffer (500mM NaCl), twice with ice cold ChIP 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. The bound proteins are eluted by incubating the beads at 65ºC for 20 minutes in 100ul of TE-SDS1%. To reverse the crosslinks the eluted material is incubated overnight at 65ºC. To clean-up the DNA, the protein are digested by adding 100ul of TE, 1ul of glycogen (20mg/ml) and 7.5ul Proteinase K (20mg/ml) and incubated for 1 hour at 65ºC. The samples are then extracted with 350uL phenol/chloroform/isoamyl alcohol (25:24:1). The DNA is then precipitated with 1 ml of 100% ethanol and 12.5uL of 5M NaCl. After an overnight incubated at -20ºC, the samples are centrifuged at maximum velocity for 1 hour at 4ºC. The supernatant is discarded and the pellet is resuspended in 100ul of water.
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).
Hybridization protocol
Resuspend Cy5 labeled IP in 4ul of water and combine with corresponding Cy3 labeled sample. Prepare the hybing solution planning to use 450ul 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.
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.
Description
Histone H3 exchange measured by the Flag-H3/Myc-H3 ratio in alpha-factor arrested cells. Flag-H3 is expressed in G1 using the GAL1 inducible promoter. Myc-H3 is expressed from its endogenous promoter.
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)). The supplementary file contains the raw GPR and TIFF files.
