title: yFR1506_MYCvsInput_CTDwt-Endo_rep1

description:
9E10 anti-Myc antibody. Anti-mouse Dynabeads (PanMouse) (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 PBS/BSA or Lysis buffer), and resuspended in 30uL PBS + 0.5% BSA / ChIP. A mix of 250uL of cell lysate + 250uL of Lysis Buffer containing protease inhibitors 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. 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.
Biological replicate 1 of 2. Rpb1-3xMYC occupancy in endo-Rpb1-3xMYC and exo-Rpb1-3xFLAG cells. CTDWT ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against MYC and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1506_MYCvsInput_CTDwt-Endo_rep1-2__S.cerevisiaeApr11_729997.bed
A genome browser-ready file for endo-CTDWT occupancy (IP/Input) in endo-RPB1-3xMYC and endo-RPB1-3xFLAG 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: yFR1506_FLAGvsInput_CTDwt-Exo_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-Flag M2 (Sigma F3165). Anti-mouse Dynabeads (PanMouse) (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. Anti-Flag M2 (Sigma F3165) is added (5ug/ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice (with PBS/BSA or Lysis buffer), and resuspended in 30uL (PBS/BSA or Lysis buffer) / ChIP. 30uL of resuspended beads/antibody is added to 500 uL of lysate, and incubated overnight at 4'C in haematological mixer. Beads are then washed in cold room (using the Dynal magnet system) twice with 1mL ice cold Lysis Buffer (no protease inhibitors), 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.
Biological replicate 1 of 2. Rpb1-3xFLAG occupancy in endo-Rpb1-3xMYC and exo-Rpb1-3xFLAG cells. CTDWT ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against FLAG and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1506_FLAGvsInput_CTDwt-Exo_rep1-2__S.cerevisiaeApr11_95606.bed
A genome browser-ready file for exo-CTDWT occupancy (IP/Input) in endo-RPB1-3xMYC and endo-RPB1-3xFLAG 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.

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:
YNB-HIS + 2%Glucose is inoculated at OD600 0.1 from an overnight preculture in the appropriate media and allowed to grow until it reaches OD600=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.
YNB-HIS + 2%Glucose is inoculated at OD600 0.1 from an overnight preculture in the appropriate media and allowed to grow until it reaches OD600=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.

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.

tag: strain

tag: genotype

value: MATa, ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3, GAL+, psi+, RPB1-3xMYC::URA3, [pFR482 (CEN, HIS3, RPB1-CTDWT-3xFLAG)]

tag: antibody

value: Flag M2 R (Sigma)


title: yFR1376_MYCvsInput_CTDwt-Endo_rep2

description:
9E10 anti-Myc antibody. Anti-mouse Dynabeads (PanMouse) (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 PBS/BSA or Lysis buffer), and resuspended in 30uL PBS + 0.5% BSA / ChIP. A mix of 250uL of cell lysate + 250uL of Lysis Buffer containing protease inhibitors 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. 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.
Biological replicate 2 of 2. Rpb1-3xMYC occupancy in endo-Rpb1-3xMYC and exo-rpb1-CTDless-3xFLAG cells. CTDWT ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against MYC and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1376_MYCvsInput_CTDwt-Endo_rep1-2__S.cerevisiaeApr11_733790.bed
A genome browser-ready file for CTDWT occupancy (IP/Input) in endo-RPB1-3xMYC and exo-rpb1-CTDless-3xFLAG 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, ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3, GAL+, psi+, RPB1-3xMYC::URA3, [pFR467 (CEN, HIS3, rpb1-CTDless-3xFLAG)]

value: 9E10 (lab stock by AF)


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. sen1-R302W-3xMYC occupancy in sen1-R302W-3xMYC cells. sen1-R302W 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 sen1-R302W gene is not tagged (Cy3-labeled).
SmthBED_yFR2565vsyFR2527_sen1-R302W-3xMYC_MYCvsnotag_2016-08-18_rep1-2__S.cerevisiaeApr11_33629.bed
A genome browser-ready file for sen1-R302W-3xMYC occupancy (IP/notag) in sen1-R302W-3xMYC 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).

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.

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.
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: RPL3_2_AB1163

type: RNA

city: Bethesda

first: Timothy

middle: G

last: Myers

state: MD

address: 50 South Drive, Room 5509

department: Research Technologies Branch

country: USA

institute: National Institute of Allergy and Infectious Diseases

description: Lentivirus KD targeting RPL3

hyb_protocol: Standard Illumina hybridization protocol

scan_protocol: Standard Illumina scanning protocol with Illumina HiScan-SQ

data_processing: Signal data was extracted from the image files with the Gene Expression module (v. 1.9.0) of the GenomeStudio software (v. 2011.1) from Illumina, Inc. Signal intensities were converted to log 2 scale. Probes that did not have detection p-value < 0.1 in at least 2 arrays were removed from the dataset.  After filtering probes, quantile normalization was applied across all arrays.

label: biotin

molecule: total RNA

organism: Homo sapiens

source_name: HEK239-Kb cells

label_protocol: Illumina TotalPrep RNA Amplification Kit (Ambion)

growth_protocol: Cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum (FBS)

extract_protocol: RNA was extracted with Trizol reagent, followed by clean-up and DNase I treatment with QIAGEN RNeasy mini kit in accordance with the prescribed protocol provided with the kit.

treatment_protocol: Transduce 293Kb cells (HEK293 cells expressing the mouse class I molecule H-2Kb from a transgene) with lentiviruses and total RNA extracted 6 days after lentivirus transduction

tag: exp


title: RPS28_2_AB1157

description: Lentivirus KD targeting RPS28


title: yFR2565vsyFR2527_sen1-R302W-3xMYC_MYCvsnotag_2016-08-18_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. sen1-R302W-3xMYC occupancy in sen1-R302W-3xMYC cells. sen1-R302W 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 sen1-R302W gene is not tagged (Cy3-labeled).
SmthBED_yFR2565vsyFR2527_sen1-R302W-3xMYC_MYCvsnotag_2016-08-18_rep1-2__S.cerevisiaeApr11_33629.bed
A genome browser-ready file for sen1-R302W-3xMYC occupancy (IP/notag) in sen1-R302W-3xMYC 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).


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 sen1-R302W-3xMYC cells. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR2565_Sen1-R302W-3xMYC_Rpb3vsInput_2016-08-18_rep1-2__S.cerevisiaeApr11_78437.bed
A genome browser-ready file for Rpb3 occupancy (IP/Input) in sen1-R302W-3xMYC 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).

value: Rpb3 (W0012 neoclone)


title: RPLP0_2_AB1159

description: Lentivirus KD targeting RPLP0


title: yFR1376_FLAGvsInput_CTDless-Exo_rep1

description:
Anti-Flag M2 (Sigma F3165). Anti-mouse Dynabeads (PanMouse) (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. Anti-Flag M2 (Sigma F3165) is added (5ug/ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice (with PBS/BSA or Lysis buffer), and resuspended in 30uL (PBS/BSA or Lysis buffer) / ChIP. 30uL of resuspended beads/antibody is added to 500 uL of lysate, and incubated overnight at 4'C in haematological mixer. Beads are then washed in cold room (using the Dynal magnet system) twice with 1mL ice cold Lysis Buffer (no protease inhibitors), 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.
Biological replicate 1 of 2. rpb1-CTDless-3xFLAG occupancy in endo-Rpb1-3xMYC and exo-rpb1-CTDless-3xFLAG cells. CTDless ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against FLAG and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1376_FLAGvsInput_CTDless-Exo_rep1-2__S.cerevisiaeApr11_171787.bed
A genome browser-ready file for CTDless occupancy (IP/Input) in endo-RPB1-3xMYC and exo-rpb1-CTDless-3xFLAG 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: yFR2564_Sen1-3xMYC_Rpb3vsInput_2016-08-18_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 WT cells. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR2564_Sen1-3xMYC_Rpb3vsInput_2016-08-18_rep1-2__S.cerevisiaeApr11_534380.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).


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. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR2564_Sen1-3xMYC_Rpb3vsInput_2016-08-18_rep1-2__S.cerevisiaeApr11_534380.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).


title: yFR1523_FLAGvsInput_CTDS7A-Exo-rep1_20140130

description:
Anti-Flag M2 (Sigma F3165). Anti-mouse Dynabeads (PanMouse) (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. Anti-Flag M2 (Sigma F3165) is added (5ug/ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice (with PBS/BSA or Lysis buffer), and resuspended in 30uL (PBS/BSA or Lysis buffer) / ChIP. 30uL of resuspended beads/antibody is added to 500 uL of lysate, and incubated overnight at 4'C in haematological mixer. Beads are then washed in cold room (using the Dynal magnet system) twice with 1mL ice cold Lysis Buffer (no protease inhibitors), 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.
Biological replicate 1 of 2. rpb1-CTDS7A-3xFLAG occupancy in endo-Rpb1-3xMYC and exo-rpb1-CTDS7A-3xFLAG cells. CTDS7A ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against FLAG and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1523_FLAGvsInput_CTDS7A-Exo-rep1-2_20140130__S.cerevisiaeApr11_954346.bed
A genome browser-ready file for CTDS7A occupancy (IP/Input) in endo-RPB1-3xMYC and exo-rpb1-CTDS7A-3xFLAG 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, ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3, GAL+, psi+, RPB1-3xMYC::URA3, [pFR494 (CEN, HIS3, rpb1-CTDS7A-3xFLAG)]


title: yFR2565_Sen1-R302W-3xMYC_Rpb3vsInput_2016-08-18_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 sen1-R302W-3xMYC cells. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR2565_Sen1-R302W-3xMYC_Rpb3vsInput_2016-08-18_rep1-2__S.cerevisiaeApr11_78437.bed
A genome browser-ready file for Rpb3 occupancy (IP/Input) in sen1-R302W-3xMYC 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).


title: SC2_1_AB1174

description: Lentivirus KD scrambled control 2


title: yFR2564vsyFR116_Sen1-3xMYC_MYCvsnotag_2016-08-18_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. Sen1-3xMYC occupancy in Sen1-3xMYC cells. Sen1 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 Sen1 gene is not tagged (Cy3-labeled).
SmthBED_yFR2564vsyFR116_Sen1-3xMYC_MYCvsnotag_2016-08-18_rep1-2__S.cerevisiaeApr11_892796.bed
A genome browser-ready file for Sen1 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).


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. Sen1-3xMYC occupancy in Sen1-3xMYC cells. Sen1 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 Sen1 gene is not tagged (Cy3-labeled).
SmthBED_yFR2564vsyFR116_Sen1-3xMYC_MYCvsnotag_2016-08-18_rep1-2__S.cerevisiaeApr11_892796.bed
A genome browser-ready file for Sen1 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).


title: yFR1508_FLAGvsInput_CTD-S2A-Exo_rep1

description:
Anti-Flag M2 (Sigma F3165). Anti-mouse Dynabeads (PanMouse) (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. Anti-Flag M2 (Sigma F3165) is added (5ug/ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice (with PBS/BSA or Lysis buffer), and resuspended in 30uL (PBS/BSA or Lysis buffer) / ChIP. 30uL of resuspended beads/antibody is added to 500 uL of lysate, and incubated overnight at 4'C in haematological mixer. Beads are then washed in cold room (using the Dynal magnet system) twice with 1mL ice cold Lysis Buffer (no protease inhibitors), 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.
Biological replicate 1 of 2. rpb1-CTDS2A-3xFLAG occupancy in endo-Rpb1-3xMYC and exo-rpb1-CTDS2A-3xFLAG cells. CTDS2A ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against FLAG and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1508_FLAGvsInput_CTD-S2A-Exo_rep1-2__S.cerevisiaeApr11_468185.bed
A genome browser-ready file for CTDS2A occupancy (IP/Input) in endo-RPB1-3xMYC and exo-rpb1-CTDS2A-3xFLAG 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, ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3, GAL+, psi+, RPB1-3xMYC::URA3, [pFR486 (CEN, HIS3, rpb1-CTDS2A-3xFLAG)]


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 nrd1DCID-3xMYC cells. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR2209_nrd1DCID-3xMYC_Rpb3vsInput_20151111_rep1-2__S.cerevisiaeApr11_844740.bed
A genome browser-ready file for Rpb3 occupancy (IP/Input) in nrd1DCID-3xMYC 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).


title: yFR1376_FLAGvsInput_CTDless-Exo_rep2

description:
Anti-Flag M2 (Sigma F3165). Anti-mouse Dynabeads (PanMouse) (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. Anti-Flag M2 (Sigma F3165) is added (5ug/ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice (with PBS/BSA or Lysis buffer), and resuspended in 30uL (PBS/BSA or Lysis buffer) / ChIP. 30uL of resuspended beads/antibody is added to 500 uL of lysate, and incubated overnight at 4'C in haematological mixer. Beads are then washed in cold room (using the Dynal magnet system) twice with 1mL ice cold Lysis Buffer (no protease inhibitors), 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.
Biological replicate 2 of 2. rpb1-CTDless-3xFLAG occupancy in endo-Rpb1-3xMYC and exo-rpb1-CTDless-3xFLAG cells. CTDless ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against FLAG and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1376_FLAGvsInput_CTDless-Exo_rep1-2__S.cerevisiaeApr11_171787.bed
A genome browser-ready file for CTDless occupancy (IP/Input) in endo-RPB1-3xMYC and exo-rpb1-CTDless-3xFLAG 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: yFR2209_nrd1DCID-3xMYC_Rpb3vsInput_20151111_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 nrd1DCID-3xMYC cells. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR2209_nrd1DCID-3xMYC_Rpb3vsInput_20151111_rep1-2__S.cerevisiaeApr11_844740.bed
A genome browser-ready file for Rpb3 occupancy (IP/Input) in nrd1DCID-3xMYC 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).


title: Mm_WT_PKHneg_msphr_L-MycER_rna_rep3

type: SRA

city: Milano

first: Pier Giuseppe

last: Pelicci

address: Via Adamello, 16

department: Department of Experimental Oncology

country: Italy

institute: European Institute of Oncology

library_source: transcriptomic

data_processing:
Basecalls performed using CASAVA version 1.9
Data were filtered using the Illumina quality control flag
Reads were aligned to the mus musculs  genome build mm10 using tophat (v2.0.8), which uses Bowtie (2.1.0) and Samtools (v0.1.18) using default parameters and --transcriptome Mus Musculus index, UCSC mm10 gtf.
Raw read counts were summarised using HTSeq (v0.5.3p9), then normalized for library size differences using estimateSizeFactors function from DESeq2 package (v1.10.1).
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include normalized expression value, log2fold change, pvalue and adjusted pvalue for each gene for each DE analysis

molecule: polyA RNA

organism: Mus musculus

source_name: Mammosphere culture

growth_protocol: cells were isolated from mammary tissue derived from WT or transgenic mice and plated in anchorage independent condition (polyHEMA coated plates) to form mammospheres. Mammospheres were serially passaged in STEM Medium (serum-free mammary epithelial basal medium MEBM, supplemented with 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin, 5 μg/ml insulin, 0.5 μg/ml hydrocortisone, 2% B27, 20 ng/ml EGF and βFGF and 4 μg/ml heparin). Mammospheres were disaggregated and RNA extracted.

extract_protocol:
RNA from mammospheres was extracted with Maxwell® 16 LEV simplyRNA cells kit (Promega) and reverse transcribed using random primers and ImProm-II™ reverse transcriptase (Promega), following manufacturer’s instructions.
Libraries of template RNA molecules were prepared from 0.5 to 2 μg high quality input RNA using the Illumina® TruSeq® RNA Sample Preparation Kit v2, following manufacturer’s instructions.

value: FVB/Hsd

tag: tissue

value: normal mammary gland

tag: age

value: 7 weeks old

value: wild type

tag: infection

value: infected with L-pWPI-MycER

tag: treatment

value: FACS sorted for PKH negative

tag: passage

value: M3 (72 hours after plating)


title: Mm_WT_PKHneg_msphr_L-MycER_rna_rep2


title: SC1_1_AB1172

description: Lentivirus KD scrambled control 1


title: RPL6_1_AB1164

description: Lentivirus KD targeting RPL6


title: RPL28_1_AB1166

description: Lentivirus KD targeting RPL28


title: yFR2209vsyFR2195_nrd1deltaCID-3xMYC_MYCvsnotag_20151111_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. nrd1DCID-3xMYC occupancy in nrd1DCID-3xMYC cells. nrd1DCID 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 nrd1DCID gene is not tagged (Cy3-labeled).
SmthBED_yFR2209vsyFR2195_nrd1deltaCID-3xMYC_MYCvsnotag_20151111_rep1-2__S.cerevisiaeApr11_650450.bed
A genome browser-ready file for nrd1DCID-3xMYC occupancy (IP/notag) in nrd1DCID-3xMYC 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).


title: Mm_WT_PKHneg_msphr_L-MycER_rna_rep1


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. nrd1DCID-3xMYC occupancy in nrd1DCID-3xMYC cells. nrd1DCID 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 nrd1DCID gene is not tagged (Cy3-labeled).
SmthBED_yFR2209vsyFR2195_nrd1deltaCID-3xMYC_MYCvsnotag_20151111_rep1-2__S.cerevisiaeApr11_650450.bed
A genome browser-ready file for nrd1DCID-3xMYC occupancy (IP/notag) in nrd1DCID-3xMYC 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).


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. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR2208_Nrd1-3xMYC_Rpb3vsInput_20151111_rep1-2__S.cerevisiaeApr11_145182.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).


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. Nrd1-3xMYC occupancy in WT cells. Nrd1 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 Nrd1 gene is not tagged (Cy3-labeled).
SmthBED_yFR2208vsyFR2194_Nrd1-3xMYC_MYCvsnotag_20151111_rep1-2__S.cerevisiaeApr11_147245.bed
A genome browser-ready file for Nrd1 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).


title: yFR2208_Nrd1-3xMYC_Rpb3vsInput_20151111_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 WT cells. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR2208_Nrd1-3xMYC_Rpb3vsInput_20151111_rep1-2__S.cerevisiaeApr11_145182.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).


title: yFR2208vsyFR2194_Nrd1-3xMYC_MYCvsnotag_20151111_rep2


title: RPLP0_1_AB1158

description: Lentivirus KD targeting RPLP0


description:
9E10 anti-Myc antibody. Anti-mouse Dynabeads (PanMouse) (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 PBS/BSA or Lysis buffer), and resuspended in 30uL PBS + 0.5% BSA / ChIP. A mix of 250uL of cell lysate + 250uL of Lysis Buffer containing protease inhibitors 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. 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. Rpb1 occupancy in endo-RPB1-3xFLAG cells. Rpb1 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against FLAG and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1308_CTDWT-3xMYC-Endo_MYCvsInput_2017-03-24_rep1-2__S.cerevisiaeApr11_91318.bed
A genome browser-ready file for Rpb1 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.

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 reaches OD600=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.
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 reaches OD600=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.


title: yFR1308_CTDWT-3xMYC-Endo_Tyr1PvsInput_2017-03-24_rep2

description:
3D12 Tyr1-P. Antibody from HelmholtzZentrum Munchen (Dirk Eick group). Protein G Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 5mL PBS + 0.5% BSA. Anti-Tyrosine 1 phosphorylated 3D12 antibody is added (5uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with 10mL Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. A mix of 250uL of cell lysate + 250uL of Lysis Buffer containing protease inhibitors 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. 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. CTDTyr1P occupancy in endo-RPB1-3xFLAG cells. CTDTyr1P ChIP sample (Cy5-labeled) was performed using a rabbit polyclonal antibody against Tyr1P and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1308_CTDWT-3xMYC-Endo_Tyr1PvsInput_2017-03-24_rep1-2__S.cerevisiaeApr11_589516.bed
A genome browser-ready file for Tyr1P 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.

value: 3D12 Tyr1-P


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 rpb1-G1097D cells. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1252_rpb1-G1097D_Rpb3vsInput_20171115_rep1-2__S.cerevisiaeApr11_240379.bed
A genome browser-ready file for Rpb3 occupancy (IP/Input) in rpb1-G1097D 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:
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 reaches OD600=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 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 reaches OD600=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 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, lys2-128∂, ura3-52, his3∆200, leu2∆0 or ∆1, trp1∆63, met15∆0, rpb1∆::CLONATMX, RPB3-3XFLAG::kanmx, [pRS315 rpb1-G1097D, CEN, LEU2]

value: MAT-alpha, lys2-128∂, ura3-52, his3∆200, leu2∆0 or ∆1, trp1∆63, met15∆0, rpb1∆::CLONATMX, RPB3-3XFLAG::kanmx, [pRS315 rpb1-G1097D, CEN, LEU2]


title: yFR1252_rpb1-G1097D_Rpb3vsInput_20171115_rep2


title: yFR1308_CTDWT-3xMYC-Endo_MYCvsInput_2017-03-24_rep2

description:
9E10 anti-Myc antibody. Anti-mouse Dynabeads (PanMouse) (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 PBS/BSA or Lysis buffer), and resuspended in 30uL PBS + 0.5% BSA / ChIP. A mix of 250uL of cell lysate + 250uL of Lysis Buffer containing protease inhibitors 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. 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. Rpb1 occupancy in endo-RPB1-3xFLAG cells. Rpb1 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against FLAG and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1308_CTDWT-3xMYC-Endo_MYCvsInput_2017-03-24_rep1-2__S.cerevisiaeApr11_91318.bed
A genome browser-ready file for Rpb1 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: RPL3_1_AB1162

description: Lentivirus KD targeting RPL3


description:
3D12 Tyr1-P. Antibody from HelmholtzZentrum Munchen (Dirk Eick group). Protein G Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 5mL PBS + 0.5% BSA. Anti-Tyrosine 1 phosphorylated 3D12 antibody is added (5uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with 10mL Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. A mix of 250uL of cell lysate + 250uL of Lysis Buffer containing protease inhibitors 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. 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. CTDTyr1P occupancy in endo-RPB1-3xFLAG cells. CTDTyr1P ChIP sample (Cy5-labeled) was performed using a rabbit polyclonal antibody against Tyr1P and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1308_CTDWT-3xMYC-Endo_Tyr1PvsInput_2017-03-24_rep1-2__S.cerevisiaeApr11_589516.bed
A genome browser-ready file for Tyr1P 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: yFR1240_WT_Rpb3vsInput_20171115_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. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1240_WT_Rpb3vsInput_20171115_rep1-2__S.cerevisiaeApr11_983099.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.

value: MATα, lys2-128∂, ura3-52, his3∆200, leu2∆0 or ∆1, trp1∆63, met15∆0, rpb1∆::CLONATMX, RPB3-3XFLAG::kanmx, [pRS315 RPB1, CEN, LEU2]

value: MAT-alpha, lys2-128∂, ura3-52, his3∆200, leu2∆0 or ∆1, trp1∆63, met15∆0, rpb1∆::CLONATMX, RPB3-3XFLAG::kanmx, [pRS315 RPB1, CEN, LEU2]


title: yFR1240_WT_Rpb3vsInput_20171115_rep1

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. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1240_WT_Rpb3vsInput_20171115_rep1-2__S.cerevisiaeApr11_983099.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: RPS28_1_AB1156

description: Lentivirus KD targeting RPS28


title: yFR1321_endoPolII_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 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.

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

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

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