1 μg/mL of rapamycin for 30 min Rpb3 ChIP. Antibody from BioLegend (665004). Pan Mouse IgG Dynabeads (50uL/ChIP) are washed twice with 10 mL PBS + 0.5% BSA (freshly made), and resuspended in 2 mL PBS + 0.5% BSA. Antibody is added (1.5 μL/ChIP), and incubated at least 5 hr at 4°C in haematological mixer. Beads are washed twice with 10 mL PBS + 0.5% BSA (freshly made), and resuspended in 30 uL PBS + 0.5% BSA/ChIP. 600 uL of lysate are added to 30 uL beads, and incubated overnight at 4°C in haematological mixer. Beads are then washed (using the Dynal magnet system) twice with 1 mL ice cold Lysis Buffer (no protease inhibitors), twice with 1 mL ice cold Lysis Buffer + additional 360mM NaCl (no protease inhibitors), twice with ice cold Wash Buffer (no protease inhibitors) and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4°C and 50 uL TE + 1% SDS is added for elution. Beads are incubated overnight at 65°C for elution and to reverse the crosslinking. Note for Inputs: whole cell extracts (WCE) are included in the protocol only at the crosslink reversal step. Add 50 uL TE + 1% SDS to 6 uL (1%) WCE and put overnight at 65°C to reverse the crosslinking. Lysis Buffer: 50 mM 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.
Growth protocol
200 mL of YPD (yeast extract-peptone-2% glucose) medium is inoculated at OD600 0.1 from an overnight preculture in the same medium. Cells are grown to an OD600 of ~0.5-0.6. Then 50 mL of cell culture is poured in a flask and treated with 1 μg/mL of rapamycin (Bio Basic Inc.) for 30 min. Culture is poured in a 50 mL tube containing 1.4 mL 37% formaldehyde for crosslinking, and incubated on rotating wheel for 30 min. Crosslinking is stopped by addition of 2.5 mL 2.5M Glycine to quench formaldehyde. Crosslinked cells are pelleted at 4°C, 3000 rpm, resuspended in 40 mL ice cold TBS and pelleted again. This wash is repeated a second time. Cell pellets are resuspended in the remaining liquid (~1 mL) and transferred to 1.5 mL screw cap tubes. Cells are pelleted by centrifugation, the supernatant removed by pipetting, and the cell pellets snap-freezed in liquid nitrogen and stored at -80°C.
Extracted molecule
genomic DNA
Extraction protocol
Cells are thawed on ice and resuspended in 700 uL Lysis buffer with protease inhibitors. 500 uL of acid-washed glass beads (Sigma) are added and the cells are lysed using a bead-beater (4 x 5 min cycles, with 5 min breaks on ice). The tubes are pierced with an 18-gauge syringe needle to allow transfer of lysate, but not beads. The pierced tubes are put in 2 mL collection tubes and spun briefly. The lysate is then homogenized by pipetting and sonicated 4x20sec at output 7 Watts, with a 1 min break between sonication cycles. The sonicated lysates are centrifuge for 5 min at 4°C max speed and the supernatant used immediately for ChIP (or saved and frozen at -80°C). 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. Beads are spun down and supernatant is transferred to a new 1.5 mL tube. 350 uL of a mix of (345 uL TE, 3 uL RNAse A 10mg/mL, 2 uL Glycogen 20mg/mL) is added to the supernatant. Samples are vortexed and incubated at 37°C for 2 hr. 15 uL 10% SDS and 7.5 uL Proteinase K are then added, vortexed and incubated for another 2 hr at 37°C. The samples are then extracted twice with 400 uL phenol/chloroform/isoamyl alcohol (25:24:1). 14 uL of 5M NaCl is added to the final extract of about 350 uL. 1 mL of cold 100% EtOH (-20°C) is added and the samples are centrifuged at maximum speed for 20 min at 4°C. The supernatant is discarded and the pellet is washed with 700 uL of cold 70% EtOH (-20°C) and centrifuged 5 min at 4°C. The supernatant is discarded and the pellet resuspended in 50 uL TE and stored at -20°C.
Label
Cy5
Label protocol
Standard Yeast aa-dUTP Labeling. Blunting: Do everything on ice. Add 70 uL of the blunting solution (11 uL NEB Buffer #2, 0.5 uL BSA 10mg/mL, 0.5 uL 20mM dNTPs, 0.2 uL T4 DNA Polymerase 3U/uL, 57.8 uL ddH2O) to 40 uL of the resuspended DNA obtained at the end of the cleanup step. For Inputs, use 4uL + 36uL ddH2O. Incubate 20 min at 12°C. Precipitation: Add 11.5 uL 3M NaOAc pH 5.2 and 0.5 uL Glycogen 20mg/mL and vortex briefly. Extract with 120 uL Phenol/Chloroform/Isoamyl alcohol (25:24:1). Transfer 110 uL to a new tube and add 230 uL of cold 100% EtOH (-20°C). Spin 20 min at 4°C. Wash the pellet with 700 uL of cold 70% EtOH (-20°C) and spin 5 min at 4°C. Resuspend the pellet in 25 uL ddH2O and place on ice. Ligation: Thaw everything and do all manipulations on ice. Add 25 uL of ligase mix (10 uL 5x T4 Ligase Buffer, 6.7 uL 15uM unidirectional linkers, 0.5 uL T4 DNA Ligase (5U/uL), 8 uL ddH2O) to the resuspended pellet from the blunting step. Incubate overnight at 16°C. Precipitation: Add 6 uL 3M NaOAc pH 5.2 and vortex. Add 130 uL of cold 100% EtOH (-20°C) and incubate at least 30 min at -20°C. Centrifuge 20 min at 4°C at maximum speed. Wash the pellet (should be a smear on the side of the tube) with 500 uL cold 70% EtOH (-20°C). Spin 5 min at 4°C. Dry the pellet and resuspend in 25 uL ddH2O. Leave on ice for about 20 min, vortex, centrifuge briefly and put on ice. Labeling: Add 15 uL of PCR labeling mix (4 uL 10x ThermoPol Buffer, 2 uL 5mM aa-dUTP mix, 1.25 uL 40uM Oligo FR1, 7.75 uL ddH2O) to the pellet from the ligation step. Start the PCR program and during the first step (4 min at 55°C), add 10 uL of the polymerase mix (1 uL 10x ThermoPol buffer, 1 uL Taq Polymerase 5U/uL, 0.01 uL Pfu Polymerase Turbo 2.5U/uL, 8 uL ddH2O). PCR cycling conditions: 1) 4 min at 55°C; 2) 5 min at 72°C; 3) 2 min at 95°C; 4) 30 sec at 95°C; 5) 30 sec at 55°C; 6) 1 min at 72°C; 7) Repeat steps 4-6 31 more times; 8) 4 min at 72°C; 9) Forever at 4°C. Purify PCR reactions with QIAquick PCR purification kit (Qiagen), following manufacturer's protocol but use a Phosphate Wash buffer and a Phosphate Elution buffer instead of their buffers PE and EB, respectively. Also, wash the columns twice and elute with 50 uL elution buffer. Speed-vac the eluate to dryness. Resuspend the pellets with fresh 4.5 uL 0.1M Sodium Carbonate buffer pH 9.0. Add 4.5 uL of the appropriate NHS-ester Cy-dye. Fresh tubes of NHS-ester Cy-dyes should be resuspended in 73 uL DMSO. Mix well and incubate at room temperature in the dark for one hour. Add 35 uL 0.1M NaOAc pH 5.2. Purify using the QIAquick PCR purification kit (Qiagen), wash twice and elute in 50 uL. Mix both samples of a pair (Cy5 and Cy3) in the same tube. Speed-vac until 10-20uL are left. Phosphate Wash buffer (100 mL): 0.5 mL 1M KPO4 pH 8.5, 15.25 mL ddH2O, 84.25 mL 95% EtOH. Solution will be cloudy upon mixing. Phosphate Elution buffer (10 mL): 0.04 mL 1M KPO4 pH 8.5, 9.96 mL ddH2O. 1M KPO4 pH 8.5 (10 mL): 9.5 mL 1M K2HPO4, 0.5 mL 1M KH2PO4. 1M Sodium Carbonate buffer pH 9.0 (100 mL): 10.8g Na2CO3, 80 mL ddH2O and pH to 9.0 with concentrated HCl. Adjust volume to 100 mL with ddH2O. Dilute 1:10 when using. Solution will change composition over time. Use only if less than a month old.
200 mL of YPD (yeast extract-peptone-2% glucose) medium is inoculated at OD600 0.1 from an overnight preculture in the same medium. Cells are grown to an OD600 of ~0.5-0.6. Then 50 mL of cell culture is poured in a flask and treated with 1 μg/mL of rapamycin (Bio Basic Inc.) for 30 min. Culture is poured in a 50 mL tube containing 1.4 mL 37% formaldehyde for crosslinking, and incubated on rotating wheel for 30 min. Crosslinking is stopped by addition of 2.5 mL 2.5M Glycine to quench formaldehyde. Crosslinked cells are pelleted at 4°C, 3000 rpm, resuspended in 40 mL ice cold TBS and pelleted again. This wash is repeated a second time. Cell pellets are resuspended in the remaining liquid (~1 mL) and transferred to 1.5 mL screw cap tubes. Cells are pelleted by centrifugation, the supernatant removed by pipetting, and the cell pellets snap-freezed in liquid nitrogen and stored at -80°C.
Extracted molecule
genomic DNA
Extraction protocol
Cells are thawed on ice and resuspended in 700 uL Lysis buffer with protease inhibitors. 500 uL of acid-washed glass beads (Sigma) are added and the cells are lysed using a bead-beater (4 x 5 min cycles, with 5 min breaks on ice). The tubes are pierced with an 18-gauge syringe needle to allow transfer of lysate, but not beads. The pierced tubes are put in 2 mL collection tubes and spun briefly. The lysate is then homogenized by pipetting and sonicated 4x20sec at output 7 Watts, with a 1 min break between sonication cycles. The sonicated lysates are centrifuge for 5 min at 4°C max speed and the supernatant used immediately for ChIP (or saved and frozen at -80°C). 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. Beads are spun down and supernatant is transferred to a new 1.5 mL tube. 350 uL of a mix of (345 uL TE, 3 uL RNAse A 10mg/mL, 2 uL Glycogen 20mg/mL) is added to the supernatant. Samples are vortexed and incubated at 37°C for 2 hr. 15 uL 10% SDS and 7.5 uL Proteinase K are then added, vortexed and incubated for another 2 hr at 37°C. The samples are then extracted twice with 400 uL phenol/chloroform/isoamyl alcohol (25:24:1). 14 uL of 5M NaCl is added to the final extract of about 350 uL. 1 mL of cold 100% EtOH (-20°C) is added and the samples are centrifuged at maximum speed for 20 min at 4°C. The supernatant is discarded and the pellet is washed with 700 uL of cold 70% EtOH (-20°C) and centrifuged 5 min at 4°C. The supernatant is discarded and the pellet resuspended in 50 uL TE and stored at -20°C.
Label
Cy3
Label protocol
Standard Yeast aa-dUTP Labeling. Blunting: Do everything on ice. Add 70 uL of the blunting solution (11 uL NEB Buffer #2, 0.5 uL BSA 10mg/mL, 0.5 uL 20mM dNTPs, 0.2 uL T4 DNA Polymerase 3U/uL, 57.8 uL ddH2O) to 40 uL of the resuspended DNA obtained at the end of the cleanup step. For Inputs, use 4uL + 36uL ddH2O. Incubate 20 min at 12°C. Precipitation: Add 11.5 uL 3M NaOAc pH 5.2 and 0.5 uL Glycogen 20mg/mL and vortex briefly. Extract with 120 uL Phenol/Chloroform/Isoamyl alcohol (25:24:1). Transfer 110 uL to a new tube and add 230 uL of cold 100% EtOH (-20°C). Spin 20 min at 4°C. Wash the pellet with 700 uL of cold 70% EtOH (-20°C) and spin 5 min at 4°C. Resuspend the pellet in 25 uL ddH2O and place on ice. Ligation: Thaw everything and do all manipulations on ice. Add 25 uL of ligase mix (10 uL 5x T4 Ligase Buffer, 6.7 uL 15uM unidirectional linkers, 0.5 uL T4 DNA Ligase (5U/uL), 8 uL ddH2O) to the resuspended pellet from the blunting step. Incubate overnight at 16°C. Precipitation: Add 6 uL 3M NaOAc pH 5.2 and vortex. Add 130 uL of cold 100% EtOH (-20°C) and incubate at least 30 min at -20°C. Centrifuge 20 min at 4°C at maximum speed. Wash the pellet (should be a smear on the side of the tube) with 500 uL cold 70% EtOH (-20°C). Spin 5 min at 4°C. Dry the pellet and resuspend in 25 uL ddH2O. Leave on ice for about 20 min, vortex, centrifuge briefly and put on ice. Labeling: Add 15 uL of PCR labeling mix (4 uL 10x ThermoPol Buffer, 2 uL 5mM aa-dUTP mix, 1.25 uL 40uM Oligo FR1, 7.75 uL ddH2O) to the pellet from the ligation step. Start the PCR program and during the first step (4 min at 55°C), add 10 uL of the polymerase mix (1 uL 10x ThermoPol buffer, 1 uL Taq Polymerase 5U/uL, 0.01 uL Pfu Polymerase Turbo 2.5U/uL, 8 uL ddH2O). PCR cycling conditions: 1) 4 min at 55°C; 2) 5 min at 72°C; 3) 2 min at 95°C; 4) 30 sec at 95°C; 5) 30 sec at 55°C; 6) 1 min at 72°C; 7) Repeat steps 4-6 31 more times; 8) 4 min at 72°C; 9) Forever at 4°C. Purify PCR reactions with QIAquick PCR purification kit (Qiagen), following manufacturer's protocol but use a Phosphate Wash buffer and a Phosphate Elution buffer instead of their buffers PE and EB, respectively. Also, wash the columns twice and elute with 50 uL elution buffer. Speed-vac the eluate to dryness. Resuspend the pellets with fresh 4.5 uL 0.1M Sodium Carbonate buffer pH 9.0. Add 4.5 uL of the appropriate NHS-ester Cy-dye. Fresh tubes of NHS-ester Cy-dyes should be resuspended in 73 uL DMSO. Mix well and incubate at room temperature in the dark for one hour. Add 35 uL 0.1M NaOAc pH 5.2. Purify using the QIAquick PCR purification kit (Qiagen), wash twice and elute in 50 uL. Mix both samples of a pair (Cy5 and Cy3) in the same tube. Speed-vac until 10-20uL are left. Phosphate Wash buffer (100 mL): 0.5 mL 1M KPO4 pH 8.5, 15.25 mL ddH2O, 84.25 mL 95% EtOH. Solution will be cloudy upon mixing. Phosphate Elution buffer (10 mL): 0.04 mL 1M KPO4 pH 8.5, 9.96 mL ddH2O. 1M KPO4 pH 8.5 (10 mL): 9.5 mL 1M K2HPO4, 0.5 mL 1M KH2PO4. 1M Sodium Carbonate buffer pH 9.0 (100 mL): 10.8g Na2CO3, 80 mL ddH2O and pH to 9.0 with concentrated HCl. Adjust volume to 100 mL 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 110 uL of hybridization mix (100 uL DIG Easy Hyb buffer, 5 uL 10mg/mL Salmon Sperm DNA, 5 uL 8mg/mL yeast tRNA). Put at 95°C for 5 min. Put at 42°C for 10 min (keep samples at 42°C while waiting for hybridization). Follow standard Agilent chamber hybridization protocol. 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 min at room temperature on an orbital shaker (cover the dish to avoid light on the slides). Wash in Wash buffer #2 pre-warmed at 31°C for 5 min 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 1 L of 5 mM Potassium phosphate buffer pH 6.6 after scanning of the slides. 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 20 to 25 min. Do not overboil it. Remove the rack and briefly rinse in a container with water. Slowly remove the rack from the liquid. 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 1 L Milli-Q water to obtain 5 mM 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 1 of 2. Rpb3 occupancy in Spt6-AA (anchor-away) cells treated with 1 μg/mL of rapamycin for 30 min. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled). SmthBED_yFR1480_Spt6-AA_Rapa30min_Rpb3vsInput_20171031_rep1-2__S.cerevisiaeApr11_395386.bed A genome browser-ready file for Rpb3 occupancy (IP/Input) in Spt6-AA (anchor-away) cells treated with 1 μg/mL of rapamycin for 30 min.
Data processing
The ChIP-chip data from individual replicates were normalized by setting the median of log2 ratios to zero. The duplicate were averaged and the data were subjected to one round of smoothing using a Gaussian sliding window with a standard deviation of 100bp to generate data points in 10bp intervals as described before (Guillemette et al., 2005, PLoS Biol 3(12):e384). Correction = Foreground-Background; Normalisation = median normalized
