50 mL of YPD (yeast extract-peptone-2% glucose, supplemented with 44 µM adenine) medium was inoculated at OD600 0.1 from an overnight preculture in the same medium and allowed to grow to an OD600 of 0.7-0.9 before crosslinking with 1% formaldehyde (Fisher Scientific, BP531-500) at room temperature for 30 min and quenched with 125 mM glycine. Crosslinked cells were collected by centrifugation and washed twice with 1X TBS (20 mM Tris-HCl pH 7.5, 150 mM NaCl). Cell pellets were snap-freezed in liquid nitrogen and stored at -80°C.
Extracted molecule
genomic DNA
Extraction protocol
IP protocol: Cell pellets were resuspended in 700 μL Lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate and protease inhibitor cocktail (1 mM PMSF, 1 mM Benzamidine, 10 μg/mL Aprotinin, 1 μg/mL Leupeptin, 1 μg/mL Pepstatin)). About the same number of OD600 units was used for all samples. Cells were lysed by bead beating and the lysate was sonicated with a Model 100 Sonic dismembrator equipped with a microprobe (Fisher Scientific), 4 x 20 sec at output 7 Watts, with a 1 min break between sonication cycles. Soluble fragmented chromatin was recovered by centrifugation. 600 μL of the chromatin sample was taken per immunoprecipitation (IP) and, when necessary, 6 μL (1%) were saved as Input sample. The following amount of antibody per IP was used: anti-Rpb3 (Neoclone W0012, 3 μL). 50 μL of Dynabeads coated with Pan Mouse IgG antibodies (Thermo Fisher Scientific, 11042) pre-coupled with the indicated antibody were added to the chromatin sample and incubated overnight at 4°C. Beads were washed twice with Lysis buffer, twice with Lysis buffer 500 (Lysis buffer + 360 mM NaCl), twice with Wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% NP40, 0.5% Na-deoxycholate, 1 mM EDTA) and once with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Input and immunoprecipitated chromatin were eluted and reverse-crosslinked with 50 μL TE/SDS (TE + 1% SDS) by incubating overnight at 65°C. Samples were treated with RNase A (345 μL TE, 3 μL 10 mg/mL RNAse A, 2 μL 20 mg/mL Glycogen) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL 20 mg/mL Proteinase K) digestion at 37°C for 2 hr. Samples were twice phenol/chloroform/isoamyl alcohol (25:24:1) extracted followed by precipitation with 200 mM NaCl and 100 % EtOH. Precipitated DNA was resuspended in 50 μL of TE before being used in ChIP-chip experiments. Cell pellets were resuspended in 700 μL Lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate and protease inhibitor cocktail (1 mM PMSF, 1 mM Benzamidine, 10 μg/mL Aprotinin, 1 μg/mL Leupeptin, 1 μg/mL Pepstatin)). About the same number of OD600 units was used for all samples. Cells were lysed by bead beating and the lysate was sonicated with a Model 100 Sonic dismembrator equipped with a microprobe (Fisher Scientific), 4 x 20 sec at output 7 Watts, with a 1 min break between sonication cycles. Soluble fragmented chromatin was recovered by centrifugation. Cleanup protocol: Beads were washed twice with Lysis buffer, twice with Lysis buffer 500 (Lysis buffer + 360 mM NaCl), twice with Wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% NP40, 0.5% Na-deoxycholate, 1 mM EDTA) and once with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Input and immunoprecipitated chromatin were eluted and reverse-crosslinked with 50 μL TE/SDS (TE + 1% SDS) by incubating overnight at 65°C. Samples were treated with RNase A (345 μL TE, 3 μL 10 mg/mL RNAse A, 2 μL 20 mg/mL Glycogen) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL 20 mg/mL Proteinase K) digestion at 37°C for 2 hr. Samples were twice phenol/chloroform/isoamyl alcohol (25:24:1) extracted followed by precipitation with 200 mM NaCl and 70% ethanol. Precipitated DNA was resuspended in 50 μL of TE before being used in ChIP-chip experiments.
Label
Cy5
Label protocol
The ChIP and Input samples were blunted as follow. 40 µL of ChIP DNA or about 400 ng of Input DNA were added to 70 µL of blunting¬¬ mix (11 µL 10X NEBuffer 2 (NEB, B7002S), 0.5 µL 10 mg/mL BSA, 0.5 µL 20 mM dNTPs and 0.2 µL (0.6 units) of T4 DNA polymerase (NEB, M0203L)) and incubated at 12˚C for 20 min. Then, the blunted DNA was extracted with phenol/chloroform/isoamyl alcohol (25:24:1) in the presence of 280 mM of NaOAc pH 5.2 and 10 μg of glycogen, precipitated with ethanol and resuspended in 25 µL ddH2O. Blunted DNA was ligated to 100 pmol of annealed linkers (see Table S2) with 2.5 units of T4 DNA ligase (Thermo Fisher Scientific, 15224041) in the supplied buffer, in a final volume of 50 µL, overnight at 16˚C. Samples were precipitated with 6 µL of 3 M NaOAc pH 5.2 and 130 µL ethanol, and resuspended in 25 µL ddH2O. 15 µL of labeling mix (4 µL 10X ThermoPol Reaction buffer (NEB, B9004S), 2 µL of 5 mM aa-dUTP/dNTP mix (5 mM each dATP, dGTP and dCTP, 3 mM dTTP and 2 mM 5-(3-aminoallyl)-dUTP (Thermo Fisher Scientific, AM8439)) and 1.25 µL of 40 µM oligo 1) were added to each sample. Samples were placed in a thermocycler and when temperature reached 55˚C, 10 µL of enzyme mix (1 µL 10X ThermoPol Reaction buffer, 1 µL (5 units) Taq DNA polymerase (Thermo Fisher Scientific, 18038240) and 0.01 µL (0.025 units) Pfu DNA polymerase (Thermo Fisher Scientific, EP0502)) were added and the following program was run. 1) 72˚C, 5 min; 2) 95˚C, 2 min; 3) 32 cycles: 95˚C, 30sec; 55˚C, 30sec; 72˚C, 1 min; 4) 72˚C, 4 min; 5) 4˚C, forever. PCR products were purified using QIAquick PCR Purification kit (QIAGEN, 28106) with few modifications: PE buffer was replaced by 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). Eluates were dried by SpeedVac and pellets were resuspended in 4.5 µL of freshly prepared 0.1 M Na2CO3 pH 9.0 buffer. 4.5 µL of Cy5 Mono-Reactive NHS Ester Fluorescent Dye (ChIP samples) or Cy3 Mono-Reactive NHS Ester Fluorescent Dye (control samples Input DNA or no-tag ChIP) (GE Healthcare, PA25001 and PA23001) were added. Samples were incubated 1 hr in the dark at room temperature. 35 µL of 0.1 M NaOAc pH 5.2 were added and samples were purified using QIAquick PCR Purification kit according to the manufacturers’ instructions.
50 mL of YPD (yeast extract-peptone-2% glucose, supplemented with 44 µM adenine) medium was inoculated at OD600 0.1 from an overnight preculture in the same medium and allowed to grow to an OD600 of 0.7-0.9 before crosslinking with 1% formaldehyde (Fisher Scientific, BP531-500) at room temperature for 30 min and quenched with 125 mM glycine. Crosslinked cells were collected by centrifugation and washed twice with 1X TBS (20 mM Tris-HCl pH 7.5, 150 mM NaCl). Cell pellets were snap-freezed in liquid nitrogen and stored at -80°C.
Extracted molecule
genomic DNA
Extraction protocol
IP protocol: none Cell pellets were resuspended in 700 μL Lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate and protease inhibitor cocktail (1 mM PMSF, 1 mM Benzamidine, 10 μg/mL Aprotinin, 1 μg/mL Leupeptin, 1 μg/mL Pepstatin)). About the same number of OD600 units was used for all samples. Cells were lysed by bead beating and the lysate was sonicated with a Model 100 Sonic dismembrator equipped with a microprobe (Fisher Scientific), 4 x 20 sec at output 7 Watts, with a 1 min break between sonication cycles. Soluble fragmented chromatin was recovered by centrifugation. Cleanup protocol: Beads were washed twice with Lysis buffer, twice with Lysis buffer 500 (Lysis buffer + 360 mM NaCl), twice with Wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% NP40, 0.5% Na-deoxycholate, 1 mM EDTA) and once with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Input and immunoprecipitated chromatin were eluted and reverse-crosslinked with 50 μL TE/SDS (TE + 1% SDS) by incubating overnight at 65°C. Samples were treated with RNase A (345 μL TE, 3 μL 10 mg/mL RNAse A, 2 μL 20 mg/mL Glycogen) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL 20 mg/mL Proteinase K) digestion at 37°C for 2 hr. Samples were twice phenol/chloroform/isoamyl alcohol (25:24:1) extracted followed by precipitation with 200 mM NaCl and 70% ethanol. Precipitated DNA was resuspended in 50 μL of TE before being used in ChIP-chip experiments.
Label
Cy3
Label protocol
The ChIP and Input samples were blunted as follow. 40 µL of ChIP DNA or about 400 ng of Input DNA were added to 70 µL of blunting¬¬ mix (11 µL 10X NEBuffer 2 (NEB, B7002S), 0.5 µL 10 mg/mL BSA, 0.5 µL 20 mM dNTPs and 0.2 µL (0.6 units) of T4 DNA polymerase (NEB, M0203L)) and incubated at 12˚C for 20 min. Then, the blunted DNA was extracted with phenol/chloroform/isoamyl alcohol (25:24:1) in the presence of 280 mM of NaOAc pH 5.2 and 10 μg of glycogen, precipitated with ethanol and resuspended in 25 µL ddH2O. Blunted DNA was ligated to 100 pmol of annealed linkers (see Table S2) with 2.5 units of T4 DNA ligase (Thermo Fisher Scientific, 15224041) in the supplied buffer, in a final volume of 50 µL, overnight at 16˚C. Samples were precipitated with 6 µL of 3 M NaOAc pH 5.2 and 130 µL ethanol, and resuspended in 25 µL ddH2O. 15 µL of labeling mix (4 µL 10X ThermoPol Reaction buffer (NEB, B9004S), 2 µL of 5 mM aa-dUTP/dNTP mix (5 mM each dATP, dGTP and dCTP, 3 mM dTTP and 2 mM 5-(3-aminoallyl)-dUTP (Thermo Fisher Scientific, AM8439)) and 1.25 µL of 40 µM oligo 1) were added to each sample. Samples were placed in a thermocycler and when temperature reached 55˚C, 10 µL of enzyme mix (1 µL 10X ThermoPol Reaction buffer, 1 µL (5 units) Taq DNA polymerase (Thermo Fisher Scientific, 18038240) and 0.01 µL (0.025 units) Pfu DNA polymerase (Thermo Fisher Scientific, EP0502)) were added and the following program was run. 1) 72˚C, 5 min; 2) 95˚C, 2 min; 3) 32 cycles: 95˚C, 30sec; 55˚C, 30sec; 72˚C, 1 min; 4) 72˚C, 4 min; 5) 4˚C, forever. PCR products were purified using QIAquick PCR Purification kit (QIAGEN, 28106) with few modifications: PE buffer was replaced by 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). Eluates were dried by SpeedVac and pellets were resuspended in 4.5 µL of freshly prepared 0.1 M Na2CO3 pH 9.0 buffer. 4.5 µL of Cy5 Mono-Reactive NHS Ester Fluorescent Dye (ChIP samples) or Cy3 Mono-Reactive NHS Ester Fluorescent Dye (control samples Input DNA or no-tag ChIP) (GE Healthcare, PA25001 and PA23001) were added. Samples were incubated 1 hr in the dark at room temperature. 35 µL of 0.1 M NaOAc pH 5.2 were added and samples were purified using QIAquick PCR Purification kit according to the manufacturers’ instructions.
Hybridization protocol
ChIP and control samples were combined and dried by SpeedVac. Combined samples were resuspended in 110 µL of fresh hybridization mix (5 µL 10 mg/mL Salmon Sperm DNA (Thermo Fisher Scientific, 15632011), 5 µL 8 mg/mL Yeast tRNA (Thermo Fisher Scientific, 15401011) and 100 µL DIG Easy Hyb (Roche, 11603558001)) and incubated 5 min at 95˚C followed by 5 min at 45˚C. Samples were hybridized to Agilent microarrays in SureHyb enabled chambers (Agilent, G2534A) with rotation at 42˚C for at least 18 hr. Microarrays were washed on an orbital shaker for 5 min at room temperature with Wash I buffer (6X SSPE, 0.005% N-Lauroylsarcosine), followed by a second wash with pre-warmed (31˚C) Wash II buffer (0.06X SSPE) for 5 min at room temperature. The microarrays used were described before (Jeronimo and Robert, 2014) and were custom designed by Agilent Technologies and contain a total of about 180,000 Tm-adjusted 60-mer probes covering the entire yeast genome with virtually no gaps between probes.
Scan protocol
Microarrays were scanned using an InnoScan900 (Innopsys) at 2 µm resolution.
Description
Biological replicate 1 of 2. RNAPII (Rpb3) occupancy by ChIP-chip 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_yFR116_WT_Rpb3vsInput_20160924_rep1-2_S.cerevisiaeApr11_857054.bed
Data processing
BED description: A genome browser-ready file for Rpb3 occupancy by ChIP-chip (IP/Input) in WT cells. BED method: The ChIP-chip data were normalized using the Limma Loess method, and replicates were combined as described previously (Ren et al. 2000, Science 290(5500):2306-9). The data were subjected to one round of smoothing using a Gaussian sliding window with a standard deviation of 100 bp to generate data points in 10-bp intervals (Guillemette et al. 2005, PLoS Biol 3(12):e384). Correction = Foreground-Background; Normalization = limma loess (subgrid)
