description:
Anti-Rpb3 (1Y26 clone). Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 5mL PBS + 0.5% BSA. Purified anti-RNAPII Rpb3 (1Y26 clone; BioLegend cat#665004) antibody is added (1.5uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. 30uL of resuspended beads/antibody is added to 400-600 uL of lysate, and incubated overnight at 4C 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), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. 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 65'C to reverse the crosslinks with the ChIP samples. 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.
none
Biological replicate 1 of 2. Rpb3 occupancy in NRD1-FRB cells treated with rapamycin for 90 min. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR2985_Nrd1-AA_rapa90min_Rpb3vsInput_20171124_rep1-2__S.cerevisiaeApr11_816845.bed
A genome browser-ready file for Rpb3 occupancy (IP/Input) in NRD1-FRB cells treated with rapamycin for 90 min.
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).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.

growth_protocol:
50 mL YNB-HIS + 2%Glucose is inoculated at OD600 0.1 from an overnight preculture in the appropriate media and allowed to grow until it reachs OD600=0.5-0.6. Cell cultures are treated with 1 microg/ml of Rapamycin and allowed to grow for additional 90 min. Culture is poured in 50mL Falcon tube containing 1.4mL 37% formaldehyde for crosslinking, and incubate on rotating wheel for 30 min. Crosslinking is stopped by addition of 2.5mL 2.5M Glycine to quench formaldehyde. Crosslinked cells are pelleted at 4'C, 3000 rpm, resuspended in 40mL ice cold TBS and pelleted again. This wash is repeated a second time. Cell pellets are resuspended in the remaining liquid (~1mL) and transfered to 1.5mL screw cap tubes. Cells are pelleted by centrifugation, the supernatant removed by pipetting, and the cells pellet snap-freeze in liquid nitrogen and stock at -80'C
50 mL YNB-HIS + 2%Glucose is inoculated at OD600 0.1 from an overnight preculture in the appropriate media and allowed to grow until it reachs OD600=0.5-0.6. Cell cultures are treated with 1 microg/ml of Rapamycin and allowed to grow for additional 90 min. Culture is poured in 50mL Falcon tube containing 1.4mL 37% formaldehyde for crosslinking, and incubate on rotating wheel for 30 min. Crosslinking is stopped by addition of 2.5mL 2.5M Glycine to quench formaldehyde. Crosslinked cells are pelleted at 4'C, 3000 rpm, resuspended in 40mL ice cold TBS and pelleted again. This wash is repeated a second time. Cell pellets are resuspended in the remaining liquid (~1mL) and transfered to 1.5mL screw cap tubes. Cells are pelleted by centrifugation, the supernatant removed by pipetting, and the cells pellet snap-freeze in liquid nitrogen and stock at -80'C

value: MATα, ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3, GAL, psi+, tor1-1, fpr1Δ::NAT, RPL13A-2xFKBP12::TRP1, NRD1-FRB::kanMX6, [pFR2 (CEN, HIS3)]

value: MAT-alpha, ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3, GAL, psi+, tor1-1, fpr1Δ::NAT, RPL13A-2xFKBP12::TRP1, NRD1-FRB::kanMX6, [pFR2 (CEN, HIS3)]


title: yFR1321_endoPolII_Rpb3vsInput_Rapamycin90min_20150328_rep1

status: Public on Jan 21 2019

type: genomic

city: Montreal

first: Francois

last: Robert

state: QC

address: 110 av des Pins Ouest

country: Canada

institute: IRCM

description:
Anti-Rpb3 (W0012). Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 5mL PBS + 0.5% BSA. W0012 antibody is added (3uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. 30uL of resuspended beads/antibody is added to 400-600 uL of lysate, and incubated overnight at 4C 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), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. 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 65'C to reverse the crosslinks with the ChIP samples. 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.
none
Biological replicate 1 of 2. Rpb3 occupancy in WT cells treated with rapamycin for 90 min. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1321_endoPOLII_Rpb3vsInput_Rapamycin90min_20150328_rep1-2__S.cerevisiaeApr11_686794.bed
A genome browser-ready file for Rpb3 occupancy (IP/Input) in WT cells treated with rapamycin for 90 min.
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).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.

hyb_protocol: Combined samples were resuspended in 110 µL of a hybridization mix (5 µL of 10 mg/mL ssDNA, 5 µL of 8 mg/mL tRNA and 100 µL DIG EASY (Roche, 11603558001)) and incubated 5 min at 95˚C followed by 5 min at 42˚C. Samples were incubated overnight on Agilent microarrays in SureHyb chambers with rotation. Microarrays were washed 5 min at room temperature with Wash I buffer (6X SSPE, 0.005% N-lauroylsarcosine) and washed a second time with a pre-warmed (42˚C) Wash II buffer (0.06X SSPE) during 5 min at room temperature.

scan_protocol: Microarrays were scanned using an InnoScan900 (Innopsys) at 2µm resolution. Scanner was set as follow: pixel size = 2µm; speed = 20; Scan Mode = Normal; Acquisition Mode = Simultaneous; Focus = Auto. Detection gain for 635 and 532 wavelengths were manually adjusted in order to have less than 5% spot saturation.

data_processing: Correction = Foreground-Background; Normalisation = limma loess (subgrid)

molecule: genomic DNA

organism: Saccharomyces cerevisiae

label_protocol: DNA blunting was done starting from 40 µL of ChIP DNA or about 400 ng of Input DNA. Samples were added to 11 µL 10X NEB2 buffer, 0.5 µL 10 mg/mL BSA, 0.5 µL 20 mM dNTPs and 0.2 µL (0.6 units) of T4 DNA pol (New England Biolabs, M0203L) in a final volume of 70 µL and incubated 20 min at 12˚C. Then, 11.5 µL of 3 M NaOAc pH 5.2 and 2 µL of 20 mg/mL glycogen were added, followed by a phenol/chloroform/isoamyl alcool extraction ethanol precipitation. DNA pellets were resuspended in 25 µL H2O and DNA linkers were ligated using 10 µL 5x ligase buffer, 6.7 µL annealed linkers (made of the annealing of the following oligos: oligo 1, GCGGTGACCCGGGAGATCTGAATTC and oligo 2, GAATTCAGATC) and 0.5 µL (2.5 units) of T4 DNA ligase (ThermoFisher, 15224041), overnight at 16˚C. Samples were precipitated with 6 µL of 3M NaOAc pH 5.2 and ethanol, and resuspended in 25 µL H2O. 15 µL of labeling mix (4 µL 10X ThermoPol buffer, 2 µL of 5 mM a.a.-dUTP/dNTP mix (5mM each dATP, dGTP and dCTP, 3mM dTTP and 2mM aminoallyl-dUTP) and 1.25 µL of oligo 1 (from 40 µM stock) was add to each sample. Samples were placed in a thermocycler and temperature was raised to 55˚C.  10 µL of enzyme mix (8 µL H2O, 1 µL 10X ThermoPoll buffer, 1 µL (5 units) Taq polymerase (ThermoFisher, 18038240) and 0.01 µL (0.025 units) Pfu (Fermentas, EP0502)) was added and the following program was run. 1) 72˚C, 5 min, 2) 95˚C, 2 min, 3) 31 cycles: 95˚C, 30sec; 55˚C, 30sec; 72˚C, 1min, 4) 72˚C, 4min, 5) 4˚C, forever. PCR products were purified using QIAgen PCR purification kit with few modifications: PE buffer was replaced by a Phosphate wash buffer (5 mM KPO4 pH 8.5 and 80% ethanol) and EB buffer was replace by Phosphate elution buffer (4 mM KPO4 pH 8.5). Supernatants were dried in a speed-vac and pellets were resuspended in 4.5 µL of freshly prepared 0.1 M Na2CO3 pH 9.0 buffer. 4.5 µL of Cy5 (ChIP samples) or Cy3 (control samples)-ester dye (GE Healthcare, CA95017-373L and CA95017-379L) were added. Samples were incubated 1 hr at room temperature. 35 µL of 0.1 M NaOAc pH 5.2 was added and samples were purified on QIAgen PCR purification kit (28106) using the manufacturer’s protocol. ChIP and control samples were combined and dried in a speed-vac.

growth_protocol:
50 mL YEP + 2%Glucose is inoculated at OD600 0.1 from an overnight preculture in the appropriate media and allowed to grow until it reachs OD600=0.5-0.6. Cell cultures are treated with 1 microg/ml of Rapamycin and allowed to grow for additional 90 min. Culture is poured in 50mL Falcon tube containing 1.4mL 37% formaldehyde for crosslinking, and incubate on rotating wheel for 30 min. Crosslinking is stopped by addition of 2.5mL 2.5M Glycine to quench formaldehyde. Crosslinked cells are pelleted at 4'C, 3000 rpm, resuspended in 40mL ice cold TBS and pelleted again. This wash is repeated a second time. Cell pellets are resuspended in the remaining liquid (~1mL) and transfered to 1.5mL screw cap tubes. Cells are pelleted by centrifugation, the supernatant removed by pipetting, and the cells pellet snap-freeze in liquid nitrogen and stock at -80'C
50 mL YEP + 2%Glucose is inoculated at OD600 0.1 from an overnight preculture in the appropriate media and allowed to grow until it reachs OD600=0.5-0.6. Cell cultures are treated with 1 microg/ml of Rapamycin and allowed to grow for additional 90 min. Culture is poured in 50mL Falcon tube containing 1.4mL 37% formaldehyde for crosslinking, and incubate on rotating wheel for 30 min. Crosslinking is stopped by addition of 2.5mL 2.5M Glycine to quench formaldehyde. Crosslinked cells are pelleted at 4'C, 3000 rpm, resuspended in 40mL ice cold TBS and pelleted again. This wash is repeated a second time. Cell pellets are resuspended in the remaining liquid (~1mL) and transfered to 1.5mL screw cap tubes. Cells are pelleted by centrifugation, the supernatant removed by pipetting, and the cells pellet snap-freeze in liquid nitrogen and stock at -80'C

extract_protocol: Frozen cell pellets were resuspended in 700 µL of Lysis buffer (50 mM HEPES-KOH 1 M pH 7.5, 140 mM NaCl, 1 mM EDTA, 1 % Triton X-100, 0.1 % Na-deoxycholate) supplemented with protease inhibitors (1 mM PMSF, 1 mM benzamidine, 10 µg/mL aprotinin, 1 µg/mL leupeptin and 1 µg/mL pepstatin) and lysed by bead beating. The extracts were sonicated with a Model 100 Sonic dismembrator equipped with a micro probe (Fisher Scientific), 4 x 20 sec at output 7 Watts, with a 1 min break between sonication cycles to obtain fragments averaging 500 bp. Samples were centrifuged at 20,000 g for 5 min at 4˚C to remove cell debris and 500 µL of soluble chromatin were used per ChIP experiment. 5 µL (1% of the amount used for IP) was also kept as an Input sample.

treatment_protocol:
1 microg/ml Rapamycin for 90 min
1 microg/ml Rapamycin for 90 min

tag: strain

tag: genotype

tag: antibody

value: Rpb3 (W0012 neoclone)

value: MAT-alpha, ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3, GAL, psi+, tor1-1, fpr1Δ::NAT, RPL13A-2xFKBP12::TRP1


title: yFR2984_WT_rapa90min_Rpb3vsInput_20171124_rep2

description:
Anti-Rpb3 (1Y26 clone). Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 5mL PBS + 0.5% BSA. Purified anti-RNAPII Rpb3 (1Y26 clone; BioLegend cat#665004) antibody is added (1.5uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. 30uL of resuspended beads/antibody is added to 400-600 uL of lysate, and incubated overnight at 4C 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), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. 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 65'C to reverse the crosslinks with the ChIP samples. 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.
none
Biological replicate 2 of 2. Rpb3 occupancy in WT cells treated with rapamycin for 90 min. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR2984_WT_rapa90min_Rpb3vsInput_20171124_rep1-2__S.cerevisiaeApr11_79916.bed
A genome browser-ready file for Rpb3 occupancy (IP/Input) in WT cells treated with rapamycin for 90 min.
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).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.

value: MAT-alpha, ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3, GAL, psi+, tor1-1, fpr1Δ::NAT, RPL13A-2xFKBP12::TRP1, [pFR2 (CEN, HIS3)]


title: yFR2985_Nrd1-AA_rapa90min_Rpb3vsInput_20171124_rep2

description:
Anti-Rpb3 (1Y26 clone). Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 5mL PBS + 0.5% BSA. Purified anti-RNAPII Rpb3 (1Y26 clone; BioLegend cat#665004) antibody is added (1.5uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. 30uL of resuspended beads/antibody is added to 400-600 uL of lysate, and incubated overnight at 4C 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), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. 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 65'C to reverse the crosslinks with the ChIP samples. 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.
none
Biological replicate 2 of 2. Rpb3 occupancy in NRD1-FRB cells treated with rapamycin for 90 min. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR2985_Nrd1-AA_rapa90min_Rpb3vsInput_20171124_rep1-2__S.cerevisiaeApr11_816845.bed
A genome browser-ready file for Rpb3 occupancy (IP/Input) in NRD1-FRB cells treated with rapamycin for 90 min.
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).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


title: yFR1894_endoPolII-GLC7-FRB_Rpb3vsInput_Rapamycin90min_20150328_rep1

description:
Anti-Rpb3 (W0012). Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 5mL PBS + 0.5% BSA. W0012 antibody is added (3uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. 30uL of resuspended beads/antibody is added to 400-600 uL of lysate, and incubated overnight at 4C 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), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. 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 65'C to reverse the crosslinks with the ChIP samples. 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.
none
Biological replicate 1 of 2. Rpb3 occupancy in GLC7-FRB cells treated with rapamycin for 90 min. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1894_endoPOLII-GLC7-FRB_Rpb3vsInput_Rapamycin90min_20150328_rep1-2__S.cerevisiaeApr11_628020.bed
A genome browser-ready file for Rpb3 occupancy (IP/Input) in GLC7-FRB cells treated with rapamycin for 90 min.
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).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.

value: MATα, ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3, GAL, psi+, tor1-1, fpr1Δ::NAT, RPL13A-2xFKBP12::TRP1, GLC7-FRB::kanMX6

value: MAT-alpha, ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3, GAL, psi+, tor1-1, fpr1Δ::NAT, RPL13A-2xFKBP12::TRP1, GLC7-FRB::kanMX6


description:
Anti-Rpb3 (W0012). Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 5mL PBS + 0.5% BSA. W0012 antibody is added (3uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. 500uL from 1 mL of cell lysate are added to 30uL beads and incubated overnight at 4C 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), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. 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 65'C to reverse the crosslinks with the ChIP samples. 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.
none
Biological replicate 1 of 2. Rpb3 occupancy in endo-rpb1-CTDS2A cells. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR2083_endoCTD-S2A_RTT103-3xMYC_Rpb3vsInput_20150626_rep1-2__S.cerevisiaeApr11_982495.bed
A genome browser-ready file for Rpb3 occupancy (IP/Input) in endo-CTDS2A cells.
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).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.

growth_protocol:
100mL YEP + 2% Glucose is inoculated at OD600 0.1 from an overnight preculture in the appropriate media and allowed to grow until it reach OD600 0.6-0.8. Culture is poured in 50mL Falcon tube containing 1.4mL 37% formaldehyde for crosslinking, and incubate on rotating wheel for 30 min. Crosslinking is stopped by addition of 2.5mL 2.5M Glycine to quench formaldehyde. Crosslinked cells are pelleted at 4'C, 3000 rpm, resuspended in 40mL ice cold TBS and pelleted again. This wash is repeated a second time. Cell pellets are resuspended in the remaining liquid (~1mL) and transferred to 1.5mL screw cap tubes. Cells are pelleted by centrifugation, the supernatant removed by pipetting, and the cells pellet snap-freeze in liquid nitrogen and stock at -80'C.
100mL YEP + 2% Glucose is inoculated at OD600 0.1 from an overnight preculture in the appropriate media and allowed to grow until it reach OD600 0.6-0.8. Culture is poured in 50mL Falcon tube containing 1.4mL 37% formaldehyde for crosslinking, and incubate on rotating wheel for 30 min. Crosslinking is stopped by addition of 2.5mL 2.5M Glycine to quench formaldehyde. Crosslinked cells are pelleted at 4'C, 3000 rpm, resuspended in 40mL ice cold TBS and pelleted again. This wash is repeated a second time. Cell pellets are resuspended in the remaining liquid (~1mL) and transferred to 1.5mL screw cap tubes. Cells are pelleted by centrifugation, the supernatant removed by pipetting, and the cells pellet snap-freeze in liquid nitrogen and stock at -80'C.

value: MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0, RPB1::rpb1-CTDS2A-KanR, RTT103-3xMYC::URA3


title: yFR2082vsyFR1773_endoCTD-WT_RTT103-3xMYC_MYCvsnotag_20150626_rep2

description:
9E10 anti-Myc antibody. Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. 9E10 anti-Myc antibody (home-prepared)  is added (10uL / ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 30uL PBS + 0.5% BSA / ChIP. 500uL from 1 mL of cell lysate are added to 30uL beads, and incubated overnight at 4'C 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 65'C to reverse the crosslinks. 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.
9E10 anti-Myc antibody. Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. 9E10 anti-Myc antibody (home-prepared)  is added (10uL / ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 30uL PBS + 0.5% BSA / ChIP. 500uL from 1 mL of cell lysate are added to 30uL beads, and incubated overnight at 4'C 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 65'C to reverse the crosslinks. 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.
Biological replicate 2 of 2. Rtt103-3xMYC occupancy in WT cells. Rtt103 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against MYC and hybridized against a similar ChIP sample performed from an isogenic strain where the Rtt103 gene is not tagged (Cy3-labeled).
SmthBED_yFR2082vsyFR1773_endoCTD-WT_RTT103-3xMYC_MYCvsnotag_20150626_rep1-2__S.cerevisiaeApr11_309129.bed
A genome browser-ready file for Rtt103 occupancy (IP/notag) in WT cells.
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).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.

value: MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0, RTT103-3xMYC::URA3

value: 9E10 (lab stock by AF)


title: yFR2083_endoCTD-S2A_RTT103-3xMYC_Rpb3vsInput_20150626_rep2

description:
Anti-Rpb3 (W0012). Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 5mL PBS + 0.5% BSA. W0012 antibody is added (3uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. 500uL from 1 mL of cell lysate are added to 30uL beads and incubated overnight at 4C 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), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. 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 65'C to reverse the crosslinks with the ChIP samples. 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.
none
Biological replicate 2 of 2. Rpb3 occupancy in endo-rpb1-CTDS2A cells. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR2083_endoCTD-S2A_RTT103-3xMYC_Rpb3vsInput_20150626_rep1-2__S.cerevisiaeApr11_982495.bed
A genome browser-ready file for Rpb3 occupancy (IP/Input) in endo-CTDS2A cells.
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).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


description:
Anti-Rpb3 (1Y26 clone). Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 5mL PBS + 0.5% BSA. Purified anti-RNAPII Rpb3 (1Y26 clone; BioLegend cat#665004) antibody is added (1.5uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. 30uL of resuspended beads/antibody is added to 400-600 uL of lysate, and incubated overnight at 4C 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), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. 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 65'C to reverse the crosslinks with the ChIP samples. 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.
none
Biological replicate 1 of 2. Rpb3 occupancy in WT cells treated with rapamycin for 90 min. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR2984_WT_rapa90min_Rpb3vsInput_20171124_rep1-2__S.cerevisiaeApr11_79916.bed
A genome browser-ready file for Rpb3 occupancy (IP/Input) in WT cells treated with rapamycin for 90 min.
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).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


description:
Anti-Rpb3 (W0012). Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 5mL PBS + 0.5% BSA. W0012 antibody is added (3uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. 500uL from 1 mL of cell lysate are added to 30uL beads and incubated overnight at 4C 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), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. 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 65'C to reverse the crosslinks with the ChIP samples. 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.
none
Biological replicate 1 of 2. Rpb3 occupancy in WT cells. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR2082_endoCTD-WT_RTT103-3xMYC_Rpb3vsInput_20150626_rep1-2__S.cerevisiaeApr11_585311.bed
A genome browser-ready file for Rpb3 occupancy (IP/Input) in WT cells.
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).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


title: yFR2082_endoCTD-WT_RTT103-3xMYC_Rpb3vsInput_20150626_rep2

description:
Anti-Rpb3 (W0012). Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 5mL PBS + 0.5% BSA. W0012 antibody is added (3uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. 500uL from 1 mL of cell lysate are added to 30uL beads and incubated overnight at 4C 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), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. 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 65'C to reverse the crosslinks with the ChIP samples. 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.
none
Biological replicate 2 of 2. Rpb3 occupancy in WT cells. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR2082_endoCTD-WT_RTT103-3xMYC_Rpb3vsInput_20150626_rep1-2__S.cerevisiaeApr11_585311.bed
A genome browser-ready file for Rpb3 occupancy (IP/Input) in WT cells.
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).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


title: yFR2083vsyFR1774_endoCTD-S2A_RTT103-3xMYC_MYCvsnotag_20150626_rep2

description:
9E10 anti-Myc antibody. Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. 9E10 anti-Myc antibody (home-prepared)  is added (10uL / ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 30uL PBS + 0.5% BSA / ChIP. 500uL from 1 mL of cell lysate are added to 30uL beads, and incubated overnight at 4'C 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 65'C to reverse the crosslinks. 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.
9E10 anti-Myc antibody. Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. 9E10 anti-Myc antibody (home-prepared)  is added (10uL / ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 30uL PBS + 0.5% BSA / ChIP. 500uL from 1 mL of cell lysate are added to 30uL beads, and incubated overnight at 4'C 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 65'C to reverse the crosslinks. 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.
Biological replicate 2 of 2. Rtt103-3xMYC occupancy in endo-rpb1-CTDS2A cells. Rtt103 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against MYC and hybridized against a similar ChIP sample performed from an isogenic strain where the Rtt103 gene is not tagged (Cy3-labeled).
SmthBED_yFR2083vsyFR1774_endoCTD-S2A_RTT103-3xMYC_MYCvsnotag_20150626_rep1-2__S.cerevisiaeApr11_6702.bed
A genome browser-ready file for Rtt103 occupancy (IP/notag) in endo-CTDS2A cells.
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).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


description:
9E10 anti-Myc antibody. Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. 9E10 anti-Myc antibody (home-prepared)  is added (10uL / ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 30uL PBS + 0.5% BSA / ChIP. 500uL from 1 mL of cell lysate are added to 30uL beads, and incubated overnight at 4'C 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 65'C to reverse the crosslinks. 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.
9E10 anti-Myc antibody. Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. 9E10 anti-Myc antibody (home-prepared)  is added (10uL / ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 30uL PBS + 0.5% BSA / ChIP. 500uL from 1 mL of cell lysate are added to 30uL beads, and incubated overnight at 4'C 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 65'C to reverse the crosslinks. 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.
Biological replicate 1 of 2. Rtt103-3xMYC occupancy in WT cells. Rtt103 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against MYC and hybridized against a similar ChIP sample performed from an isogenic strain where the Rtt103 gene is not tagged (Cy3-labeled).
SmthBED_yFR2082vsyFR1773_endoCTD-WT_RTT103-3xMYC_MYCvsnotag_20150626_rep1-2__S.cerevisiaeApr11_309129.bed
A genome browser-ready file for Rtt103 occupancy (IP/notag) in WT cells.
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).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


description:
9E10 anti-Myc antibody. Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. 9E10 anti-Myc antibody (home-prepared)  is added (10uL / ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 30uL PBS + 0.5% BSA / ChIP. 500uL from 1 mL of cell lysate are added to 30uL beads, and incubated overnight at 4'C 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 65'C to reverse the crosslinks. 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.
9E10 anti-Myc antibody. Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. 9E10 anti-Myc antibody (home-prepared)  is added (10uL / ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 30uL PBS + 0.5% BSA / ChIP. 500uL from 1 mL of cell lysate are added to 30uL beads, and incubated overnight at 4'C 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 65'C to reverse the crosslinks. 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.
Biological replicate 1 of 2. Pcf11-3xMYC occupancy in endo-rpb1-CTDS2A cells. Pcf11 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against MYC and hybridized against a similar ChIP sample performed from an isogenic strain where the PCF11 gene is not tagged (Cy3-labeled).
SmthBED_yFR1918vsyFR1774_endoCTD-S2A_PCF11-3xMYC_MYCvsnotag_20150626_rep1-2__S.cerevisiaeApr11_290908.bed
A genome browser-ready file for Pcf11 occupancy (IP/notag) in endo-CTDS2A cells.
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).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.

value: MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0, RPB1::rpb1-CTDS2A-KanR, PCF11-3xMYC::URA3


title: yFR1917vsyFR1773_endoCTD-WT_PCF11-3xMYC_MYCvsnotag_20150626_rep2

description:
9E10 anti-Myc antibody. Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. 9E10 anti-Myc antibody (home-prepared)  is added (10uL / ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 30uL PBS + 0.5% BSA / ChIP. 500uL from 1 mL of cell lysate are added to 30uL beads, and incubated overnight at 4'C 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 65'C to reverse the crosslinks. 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.
9E10 anti-Myc antibody. Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. 9E10 anti-Myc antibody (home-prepared)  is added (10uL / ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 30uL PBS + 0.5% BSA / ChIP. 500uL from 1 mL of cell lysate are added to 30uL beads, and incubated overnight at 4'C 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 65'C to reverse the crosslinks. 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.
Biological replicate 2 of 2. Pcf11-3xMYC occupancy in WT cells. Pcf11 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against MYC and hybridized against a similar ChIP sample performed from an isogenic strain where the PCF11 gene is not tagged (Cy3-labeled).
SmthBED_yFR1917vsyFR1773_endoCTD-WT_PCF11-3xMYC_MYCvsnotag_20150626_rep1-2__S.cerevisiaeApr11_665236.bed
A genome browser-ready file for Pcf11 occupancy (IP/notag) in WT cells.
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).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.

value: MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0, PCF11-3xMYC::URA3


title: yFR1918_endoCTD-S2A_PCF11-3xMYC_Rpb3vsInput_20150626_rep2

description:
Anti-Rpb3 (W0012). Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 5mL PBS + 0.5% BSA. W0012 antibody is added (3uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. 500uL from 1 mL of cell lysate are added to 30uL beads and incubated overnight at 4C 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), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. 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 65'C to reverse the crosslinks with the ChIP samples. 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.
none
Biological replicate 2 of 2. Rpb3 occupancy in endo-rpb1-CTDS2A cells. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1918_endoCTD-S2A_PCF11-3xMYC_Rpb3vsInput_20150626_rep1-2__S.cerevisiaeApr11_543005.bed
A genome browser-ready file for Rpb3 occupancy (IP/Input) in endo-CTDS2A cells.
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).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


description:
Anti-Rpb3 (W0012). Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 5mL PBS + 0.5% BSA. W0012 antibody is added (3uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. 500uL from 1 mL of cell lysate are added to 30uL beads and incubated overnight at 4C 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), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. 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 65'C to reverse the crosslinks with the ChIP samples. 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.
none
Biological replicate 1 of 2. Rpb3 occupancy in endo-rpb1-CTDS2A cells. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1918_endoCTD-S2A_PCF11-3xMYC_Rpb3vsInput_20150626_rep1-2__S.cerevisiaeApr11_543005.bed
A genome browser-ready file for Rpb3 occupancy (IP/Input) in endo-CTDS2A cells.
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).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


title: yFR1894_endoPolII-GLC7-FRB_Rpb3vsInput_Rapamycin90min_20150328_rep2

description:
Anti-Rpb3 (W0012). Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 5mL PBS + 0.5% BSA. W0012 antibody is added (3uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. 30uL of resuspended beads/antibody is added to 400-600 uL of lysate, and incubated overnight at 4C 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), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. 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 65'C to reverse the crosslinks with the ChIP samples. 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.
none
Biological replicate 2 of 2. Rpb3 occupancy in GLC7-FRB cells treated with rapamycin for 90 min. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1894_endoPOLII-GLC7-FRB_Rpb3vsInput_Rapamycin90min_20150328_rep1-2__S.cerevisiaeApr11_628020.bed
A genome browser-ready file for Rpb3 occupancy (IP/Input) in GLC7-FRB cells treated with rapamycin for 90 min.
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).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


description:
Anti-Rpb3 (W0012). Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 5mL PBS + 0.5% BSA. W0012 antibody is added (3uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. 500uL from 1 mL of cell lysate are added to 30uL beads and incubated overnight at 4C 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), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. 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 65'C to reverse the crosslinks with the ChIP samples. 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.
none
Biological replicate 1 of 2. Rpb3 occupancy in WT cells. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1917_endoCTD-WT_PCF11-3xMYC_Rpb3vsInput_20150626_rep1-2__S.cerevisiaeApr11_585232.bed
A genome browser-ready file for Rpb3 occupancy (IP/Input) in WT cells.
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).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


description:
9E10 anti-Myc antibody. Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. 9E10 anti-Myc antibody (home-prepared)  is added (10uL / ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 30uL PBS + 0.5% BSA / ChIP. 500uL from 1 mL of cell lysate are added to 30uL beads, and incubated overnight at 4'C 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 65'C to reverse the crosslinks. 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.
9E10 anti-Myc antibody. Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. 9E10 anti-Myc antibody (home-prepared)  is added (10uL / ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 30uL PBS + 0.5% BSA / ChIP. 500uL from 1 mL of cell lysate are added to 30uL beads, and incubated overnight at 4'C 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 65'C to reverse the crosslinks. 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.
Biological replicate 1 of 2. Pcf11-3xMYC occupancy in WT cells. Pcf11 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against MYC and hybridized against a similar ChIP sample performed from an isogenic strain where the PCF11 gene is not tagged (Cy3-labeled).
SmthBED_yFR1917vsyFR1773_endoCTD-WT_PCF11-3xMYC_MYCvsnotag_20150626_rep1-2__S.cerevisiaeApr11_665236.bed
A genome browser-ready file for Pcf11 occupancy (IP/notag) in WT cells.
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).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


title: yFR1918vsyFR1774_endoCTD-S2A_PCF11-3xMYC_MYCvsnotag_20150626_rep2

description:
9E10 anti-Myc antibody. Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. 9E10 anti-Myc antibody (home-prepared)  is added (10uL / ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 30uL PBS + 0.5% BSA / ChIP. 500uL from 1 mL of cell lysate are added to 30uL beads, and incubated overnight at 4'C 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 65'C to reverse the crosslinks. 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.
9E10 anti-Myc antibody. Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. 9E10 anti-Myc antibody (home-prepared)  is added (10uL / ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 30uL PBS + 0.5% BSA / ChIP. 500uL from 1 mL of cell lysate are added to 30uL beads, and incubated overnight at 4'C 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 65'C to reverse the crosslinks. 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.
Biological replicate 2 of 2. Pcf11-3xMYC occupancy in endo-rpb1-CTDS2A cells. Pcf11 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against MYC and hybridized against a similar ChIP sample performed from an isogenic strain where the PCF11 gene is not tagged (Cy3-labeled).
SmthBED_yFR1918vsyFR1774_endoCTD-S2A_PCF11-3xMYC_MYCvsnotag_20150626_rep1-2__S.cerevisiaeApr11_290908.bed
A genome browser-ready file for Pcf11 occupancy (IP/notag) in endo-CTDS2A cells.
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).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


description:
9E10 anti-Myc antibody. Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. 9E10 anti-Myc antibody (home-prepared)  is added (10uL / ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 30uL PBS + 0.5% BSA / ChIP. 500uL from 1 mL of cell lysate are added to 30uL beads, and incubated overnight at 4'C 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 65'C to reverse the crosslinks. 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.
9E10 anti-Myc antibody. Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. 9E10 anti-Myc antibody (home-prepared)  is added (10uL / ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 30uL PBS + 0.5% BSA / ChIP. 500uL from 1 mL of cell lysate are added to 30uL beads, and incubated overnight at 4'C 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 65'C to reverse the crosslinks. 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.
Biological replicate 1 of 2. Rtt103-3xMYC occupancy in WT cells. Rtt103 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against MYC and hybridized against a similar ChIP sample performed from an isogenic strain where the Rtt103 gene is not tagged (Cy3-labeled).
SmthBED_yFR1983vsyFR117_WT_RTT103-3xMYC_MYCvsnotag_20150413_rep1-2__S.cerevisiaeApr11_300631.bed
A genome browser-ready file for Rtt103 occupancy (IP/notag) in WT cells.
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).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


description:
Anti-Rpb3 (W0012). Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 5mL PBS + 0.5% BSA. W0012 antibody is added (3uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. 500uL from 1 mL of cell lysate are added to 30uL beads and incubated overnight at 4C 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), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. 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 65'C to reverse the crosslinks with the ChIP samples. 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.
none
Biological replicate 1 of 2. Rpb3 occupancy in ctk1D cells. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1970_ctk1delta_RTT103-3xMYC_Rpb3vsInput_20150413_rep1-2__S.cerevisiaeApr11_523681.bed
A genome browser-ready file for Rpb3 occupancy (IP/Input) in ctk1Δ cells.
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).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.

value: MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0, ctk1Δ::KanMX4, RTT103-3xMYC::URA3


title: yFR1970_ctk1delta_RTT103-3xMYC_Rpb3vsInput_20150413_rep2

description:
Anti-Rpb3 (W0012). Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 5mL PBS + 0.5% BSA. W0012 antibody is added (3uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. 500uL from 1 mL of cell lysate are added to 30uL beads and incubated overnight at 4C 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), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. 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 65'C to reverse the crosslinks with the ChIP samples. 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.
none
Biological replicate 2 of 2. Rpb3 occupancy in ctk1D cells. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1970_ctk1delta_RTT103-3xMYC_Rpb3vsInput_20150413_rep1-2__S.cerevisiaeApr11_523681.bed
A genome browser-ready file for Rpb3 occupancy (IP/Input) in ctk1Δ cells.
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).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


title: yFR1983vsyFR117_WT_RTT103-3xMYC_MYCvsnotag_20150413_rep2

description:
9E10 anti-Myc antibody. Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. 9E10 anti-Myc antibody (home-prepared)  is added (10uL / ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 30uL PBS + 0.5% BSA / ChIP. 500uL from 1 mL of cell lysate are added to 30uL beads, and incubated overnight at 4'C 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 65'C to reverse the crosslinks. 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.
9E10 anti-Myc antibody. Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. 9E10 anti-Myc antibody (home-prepared)  is added (10uL / ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 30uL PBS + 0.5% BSA / ChIP. 500uL from 1 mL of cell lysate are added to 30uL beads, and incubated overnight at 4'C 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 65'C to reverse the crosslinks. 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.
Biological replicate 2 of 2. Rtt103-3xMYC occupancy in WT cells. Rtt103 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against MYC and hybridized against a similar ChIP sample performed from an isogenic strain where the Rtt103 gene is not tagged (Cy3-labeled).
SmthBED_yFR1983vsyFR117_WT_RTT103-3xMYC_MYCvsnotag_20150413_rep1-2__S.cerevisiaeApr11_300631.bed
A genome browser-ready file for Rtt103 occupancy (IP/notag) in WT cells.
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).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


title: yFR1970vsyFR551_ctk1delta_RTT103-3xMYC_MYCvsnotag_20150413_rep2

description:
9E10 anti-Myc antibody. Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. 9E10 anti-Myc antibody (home-prepared)  is added (10uL / ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 30uL PBS + 0.5% BSA / ChIP. 500uL from 1 mL of cell lysate are added to 30uL beads, and incubated overnight at 4'C 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 65'C to reverse the crosslinks. 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.
9E10 anti-Myc antibody. Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. 9E10 anti-Myc antibody (home-prepared)  is added (10uL / ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 30uL PBS + 0.5% BSA / ChIP. 500uL from 1 mL of cell lysate are added to 30uL beads, and incubated overnight at 4'C 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 65'C to reverse the crosslinks. 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.
Biological replicate 2 of 2. Rtt103-3xMYC occupancy in ctk1D cells. Rtt103 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against MYC and hybridized against a similar ChIP sample performed from an isogenic strain where the Rtt103 gene is not tagged (Cy3-labeled).
SmthBED_yFR1970vsyFR551_ctk1delta_RTT103-3xMYC_MYCvsnotag_20150413_rep1-2__S.cerevisiaeApr11_349466.bed
A genome browser-ready file for Rtt103 occupancy (IP/notag) in ctk1Δ cells.
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).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


description:
Anti-Rpb3 (W0012). Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 5mL PBS + 0.5% BSA. W0012 antibody is added (3uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. 500uL from 1 mL of cell lysate are added to 30uL beads and incubated overnight at 4C 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), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. 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 65'C to reverse the crosslinks with the ChIP samples. 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.
none
Biological replicate 1 of 2. Rpb3 occupancy in WT cells. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1983_WT_RTT103-3xMYC_Rpb3vsInput_20150413_rep1-2__S.cerevisiaeApr11_598966.bed
A genome browser-ready file for Rpb3 occupancy (IP/Input) in WT cells.
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).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


title: yFR1983_WT_RTT103-3xMYC_Rpb3vsInput_20150413_rep2

description:
Anti-Rpb3 (W0012). Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 5mL PBS + 0.5% BSA. W0012 antibody is added (3uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. 500uL from 1 mL of cell lysate are added to 30uL beads and incubated overnight at 4C 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), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. 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 65'C to reverse the crosslinks with the ChIP samples. 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.
none
Biological replicate 2 of 2. Rpb3 occupancy in WT cells. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1983_WT_RTT103-3xMYC_Rpb3vsInput_20150413_rep1-2__S.cerevisiaeApr11_598966.bed
A genome browser-ready file for Rpb3 occupancy (IP/Input) in WT cells.
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).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


description:
9E10 anti-Myc antibody. Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. 9E10 anti-Myc antibody (home-prepared)  is added (10uL / ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 30uL PBS + 0.5% BSA / ChIP. 500uL from 1 mL of cell lysate are added to 30uL beads, and incubated overnight at 4'C 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 65'C to reverse the crosslinks. 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.
9E10 anti-Myc antibody. Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. 9E10 anti-Myc antibody (home-prepared)  is added (10uL / ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 30uL PBS + 0.5% BSA / ChIP. 500uL from 1 mL of cell lysate are added to 30uL beads, and incubated overnight at 4'C 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 65'C to reverse the crosslinks. 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.
Biological replicate 1 of 2. Rtt103-3xMYC occupancy in endo-rpb1-CTDS2A cells. Rtt103 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against MYC and hybridized against a similar ChIP sample performed from an isogenic strain where the Rtt103 gene is not tagged (Cy3-labeled).
SmthBED_yFR2083vsyFR1774_endoCTD-S2A_RTT103-3xMYC_MYCvsnotag_20150626_rep1-2__S.cerevisiaeApr11_6702.bed
A genome browser-ready file for Rtt103 occupancy (IP/notag) in endo-CTDS2A cells.
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).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


description:
Anti-Rpb3 (W0012). Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 5mL PBS + 0.5% BSA. W0012 antibody is added (3uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. 500uL from 1 mL of cell lysate are added to 30uL beads and incubated overnight at 4C 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), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. 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 65'C to reverse the crosslinks with the ChIP samples. 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.
none
Biological replicate 1 of 2. Rpb3 occupancy in ctk1D cells. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1969_ctk1delta_PCF11-3xMYC_Rpb3vsInput_20150413_rep1-2__S.cerevisiaeApr11_292519.bed
A genome browser-ready file for Rpb3 occupancy (IP/Input) in ctk1Δ cells.
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).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.

value: MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0, ctk1Δ::KanMX4, PCF11-3xMYC::URA3