ERS5551480 SAMEA7804227 4932 Saccharomyces cerevisiae Protocols: Yeast cells were collected by centrifugation 10min at 1000x g, washed twice with sterilized water, and resuspended into selective media Strain YPH499 (Mata ura3-52 lys2-801 ade2-101 trp1-Δ63 his3-Δ200 leu2-Δ1) was transformed with 2, 3 or 4 expression plasmids by the standard lithium acetate procedure. Transformants were selected on plates of appropriate selective medium with 2% Raffinose and 10µg/ml doxycycline to repress any expression. Selected transformants (2 to 4 transformants per combination of plasmids) were grown on selective liquid medium with 2% Raffinose and 10µg/ml doxycycline up to OD600=0.5. Then, yeast cells were spun 10min at 1000x g, washed twice with sterilized water, and resuspended into selective media with 1% Raffinose and 2% Galactose without doxycycline to allow expression of all four DNMTs. For experiments on synchronized cells, cells were treated with alpha-factor (3µM final) for 2 to 4 hours to synchronise cells in G1 DNA was purified using phenol-chloroform extraction and concentrated by ethanol precipitation. Each chromatin extract was then used as an input control and for two separate immunoprecipitations (H3K4me1 and H4K4me3), thus INE1 is the input both for the M1E1 and M3E1, INE2 for the M1E2 and M3E2, INM1 for M1M1 and M3M1 and INM2 for M1M2 and M3M2. After crosslinking, spheroplasts were isolated as described before using zymolase and resuspended in 0.3 ml of lysis buffer (50 mM Hepes-KOH at pH 7.2, 140 mM NaCl, 1 mM EDTA, 0.1% Deoxycholic acid sodium salt and 1% Triton X-100) containing a cocktail of protease inhibitors (EDTA-free Tablets, Roche). An equal volume of glass beads (0.5-mm diameter) was added, and the spheroplasts were broken using a bead-beater (FastPrep-24, Biomedicals). Glass beads were then removed and the lysate was transferred to a Sorenson tubes to digest the chromatin into fragments of 300 nucleotides using the Bioruptor Pico (30 cycles, 30”on/30”off). The whole extract was clarified by centrifugation for 10 min at 5000 × g at 4 °C and an aliquot was taken as input. In parallel, 50 µl of Dynabeads M-280 Sheep anti Rabbit IgG (Thermo Fisher) per sample were washed twice with PBS+5mg/ml of BSA and incubated overnight at 4ºC with 2.5 µg of the primary antibody (H3K4me1 (ab8895, Abcam) or H3K4me3 (ab8580, Abcam)). Beads were then washed with PBS+5mg/ml of BSA and resuspended in 30 µl/sample of PBS-BSA 5mg/ml. Extracts were then incubated 2h at 4ºC with the Dynabeads, previously conjugated with the primary antibody, and then washed two times with lysis buffer, two times with the lysis buffer supplemented with 360mM NaCl,, 2 times with washing buffer (0.5% Deoxycholic acid sodium salt, 10mM TRIS pH8, 250 mM LiCl, 0.5% NP-40, 1 mM EDTA) and one time with TE (10Mm Tris-HCl pH 7.5,1mM EDTA). Dynabeads were resuspended in 80 µl of Elution Buffer (50 mM TRIS, 10 mM of EDTA, 1% SDS) and crosslinking was reverted incubating overnight at 65ºC followed by 2h incubation at 37ºC with 0.80 mg/ml per sample of proteinase K. ENA first public 2021-03-10 ENA last update 2021-01-12 External Id SAMEA7804227 INSDC center alias Institute for Research in Biomedicine (IRB Barcelona)-The Barcelona Institute of Science and Technology. INSDC center name Institute for Research in Biomedicine (IRB Barcelona)-The Barcelona Institute of Science and Technology. INSDC first public 2021-03-10T04:08:23Z INSDC last update 2021-01-12T20:09:29Z INSDC status public Submitter Id E-MTAB-10001:INE1 broker name ArrayExpress common name baker's yeast genetic modification empty vector genotype Mata ura3-52 lys2-801 ade2-101 trp1-63 his3-200 leu2-1 growth condition Raffinose/Galactose phenotype wild type sample name E-MTAB-10001:INE1 strain YPH499 ERS5551481 SAMEA7804228 4932 Saccharomyces cerevisiae Protocols: Yeast cells were collected by centrifugation 10min at 1000x g, washed twice with sterilized water, and resuspended into selective media Strain YPH499 (Mata ura3-52 lys2-801 ade2-101 trp1-Δ63 his3-Δ200 leu2-Δ1) was transformed with 2, 3 or 4 expression plasmids by the standard lithium acetate procedure. Transformants were selected on plates of appropriate selective medium with 2% Raffinose and 10µg/ml doxycycline to repress any expression. Selected transformants (2 to 4 transformants per combination of plasmids) were grown on selective liquid medium with 2% Raffinose and 10µg/ml doxycycline up to OD600=0.5. Then, yeast cells were spun 10min at 1000x g, washed twice with sterilized water, and resuspended into selective media with 1% Raffinose and 2% Galactose without doxycycline to allow expression of all four DNMTs. For experiments on synchronized cells, cells were treated with alpha-factor (3µM final) for 2 to 4 hours to synchronise cells in G1 DNA was purified using phenol-chloroform extraction and concentrated by ethanol precipitation. Each chromatin extract was then used as an input control and for two separate immunoprecipitations (H3K4me1 and H4K4me3), thus INE1 is the input both for the M1E1 and M3E1, INE2 for the M1E2 and M3E2, INM1 for M1M1 and M3M1 and INM2 for M1M2 and M3M2. After crosslinking, spheroplasts were isolated as described before using zymolase and resuspended in 0.3 ml of lysis buffer (50 mM Hepes-KOH at pH 7.2, 140 mM NaCl, 1 mM EDTA, 0.1% Deoxycholic acid sodium salt and 1% Triton X-100) containing a cocktail of protease inhibitors (EDTA-free Tablets, Roche). An equal volume of glass beads (0.5-mm diameter) was added, and the spheroplasts were broken using a bead-beater (FastPrep-24, Biomedicals). Glass beads were then removed and the lysate was transferred to a Sorenson tubes to digest the chromatin into fragments of 300 nucleotides using the Bioruptor Pico (30 cycles, 30”on/30”off). The whole extract was clarified by centrifugation for 10 min at 5000 × g at 4 °C and an aliquot was taken as input. In parallel, 50 µl of Dynabeads M-280 Sheep anti Rabbit IgG (Thermo Fisher) per sample were washed twice with PBS+5mg/ml of BSA and incubated overnight at 4ºC with 2.5 µg of the primary antibody (H3K4me1 (ab8895, Abcam) or H3K4me3 (ab8580, Abcam)). Beads were then washed with PBS+5mg/ml of BSA and resuspended in 30 µl/sample of PBS-BSA 5mg/ml. Extracts were then incubated 2h at 4ºC with the Dynabeads, previously conjugated with the primary antibody, and then washed two times with lysis buffer, two times with the lysis buffer supplemented with 360mM NaCl,, 2 times with washing buffer (0.5% Deoxycholic acid sodium salt, 10mM TRIS pH8, 250 mM LiCl, 0.5% NP-40, 1 mM EDTA) and one time with TE (10Mm Tris-HCl pH 7.5,1mM EDTA). Dynabeads were resuspended in 80 µl of Elution Buffer (50 mM TRIS, 10 mM of EDTA, 1% SDS) and crosslinking was reverted incubating overnight at 65ºC followed by 2h incubation at 37ºC with 0.80 mg/ml per sample of proteinase K. ENA first public 2021-03-10 ENA last update 2021-01-12 External Id SAMEA7804228 INSDC center alias Institute for Research in Biomedicine (IRB Barcelona)-The Barcelona Institute of Science and Technology. INSDC center name Institute for Research in Biomedicine (IRB Barcelona)-The Barcelona Institute of Science and Technology. INSDC first public 2021-03-10T04:08:23Z INSDC last update 2021-01-12T20:09:29Z INSDC status public Submitter Id E-MTAB-10001:INE2 broker name ArrayExpress common name baker's yeast genetic modification empty vector genotype Mata ura3-52 lys2-801 ade2-101 trp1-63 his3-200 leu2-1 growth condition Raffinose/Galactose phenotype wild type sample name E-MTAB-10001:INE2 strain YPH499 ERS5551482 SAMEA7804229 4932 Saccharomyces cerevisiae Protocols: Yeast cells were collected by centrifugation 10min at 1000x g, washed twice with sterilized water, and resuspended into selective media Strain YPH499 (Mata ura3-52 lys2-801 ade2-101 trp1-Δ63 his3-Δ200 leu2-Δ1) was transformed with 2, 3 or 4 expression plasmids by the standard lithium acetate procedure. Transformants were selected on plates of appropriate selective medium with 2% Raffinose and 10µg/ml doxycycline to repress any expression. Selected transformants (2 to 4 transformants per combination of plasmids) were grown on selective liquid medium with 2% Raffinose and 10µg/ml doxycycline up to OD600=0.5. Then, yeast cells were spun 10min at 1000x g, washed twice with sterilized water, and resuspended into selective media with 1% Raffinose and 2% Galactose without doxycycline to allow expression of all four DNMTs. For experiments on synchronized cells, cells were treated with alpha-factor (3µM final) for 2 to 4 hours to synchronise cells in G1 DNA was purified using phenol-chloroform extraction and concentrated by ethanol precipitation. Each chromatin extract was then used as an input control and for two separate immunoprecipitations (H3K4me1 and H4K4me3), thus INE1 is the input both for the M1E1 and M3E1, INE2 for the M1E2 and M3E2, INM1 for M1M1 and M3M1 and INM2 for M1M2 and M3M2. After crosslinking, spheroplasts were isolated as described before using zymolase and resuspended in 0.3 ml of lysis buffer (50 mM Hepes-KOH at pH 7.2, 140 mM NaCl, 1 mM EDTA, 0.1% Deoxycholic acid sodium salt and 1% Triton X-100) containing a cocktail of protease inhibitors (EDTA-free Tablets, Roche). An equal volume of glass beads (0.5-mm diameter) was added, and the spheroplasts were broken using a bead-beater (FastPrep-24, Biomedicals). Glass beads were then removed and the lysate was transferred to a Sorenson tubes to digest the chromatin into fragments of 300 nucleotides using the Bioruptor Pico (30 cycles, 30”on/30”off). The whole extract was clarified by centrifugation for 10 min at 5000 × g at 4 °C and an aliquot was taken as input. In parallel, 50 µl of Dynabeads M-280 Sheep anti Rabbit IgG (Thermo Fisher) per sample were washed twice with PBS+5mg/ml of BSA and incubated overnight at 4ºC with 2.5 µg of the primary antibody (H3K4me1 (ab8895, Abcam) or H3K4me3 (ab8580, Abcam)). Beads were then washed with PBS+5mg/ml of BSA and resuspended in 30 µl/sample of PBS-BSA 5mg/ml. Extracts were then incubated 2h at 4ºC with the Dynabeads, previously conjugated with the primary antibody, and then washed two times with lysis buffer, two times with the lysis buffer supplemented with 360mM NaCl,, 2 times with washing buffer (0.5% Deoxycholic acid sodium salt, 10mM TRIS pH8, 250 mM LiCl, 0.5% NP-40, 1 mM EDTA) and one time with TE (10Mm Tris-HCl pH 7.5,1mM EDTA). Dynabeads were resuspended in 80 µl of Elution Buffer (50 mM TRIS, 10 mM of EDTA, 1% SDS) and crosslinking was reverted incubating overnight at 65ºC followed by 2h incubation at 37ºC with 0.80 mg/ml per sample of proteinase K. ENA first public 2021-03-10 ENA last update 2021-01-12 External Id SAMEA7804229 INSDC center alias Institute for Research in Biomedicine (IRB Barcelona)-The Barcelona Institute of Science and Technology. INSDC center name Institute for Research in Biomedicine (IRB Barcelona)-The Barcelona Institute of Science and Technology. INSDC first public 2021-03-10T04:08:23Z INSDC last update 2021-01-12T20:09:29Z INSDC status public Submitter Id E-MTAB-10001:INM1 broker name ArrayExpress common name baker's yeast genetic modification vector expressing DNMT1, 3a, 3b, 3L genotype Mata ura3-52 lys2-801 ade2-101 trp1-63 his3-200 leu2-1 growth condition Raffinose/Galactose phenotype DNA methylation sample name E-MTAB-10001:INM1 stimulus doxycycline induction strain YPH499 ERS5551483 SAMEA7804230 4932 Saccharomyces cerevisiae Protocols: Yeast cells were collected by centrifugation 10min at 1000x g, washed twice with sterilized water, and resuspended into selective media Strain YPH499 (Mata ura3-52 lys2-801 ade2-101 trp1-Δ63 his3-Δ200 leu2-Δ1) was transformed with 2, 3 or 4 expression plasmids by the standard lithium acetate procedure. Transformants were selected on plates of appropriate selective medium with 2% Raffinose and 10µg/ml doxycycline to repress any expression. Selected transformants (2 to 4 transformants per combination of plasmids) were grown on selective liquid medium with 2% Raffinose and 10µg/ml doxycycline up to OD600=0.5. Then, yeast cells were spun 10min at 1000x g, washed twice with sterilized water, and resuspended into selective media with 1% Raffinose and 2% Galactose without doxycycline to allow expression of all four DNMTs. For experiments on synchronized cells, cells were treated with alpha-factor (3µM final) for 2 to 4 hours to synchronise cells in G1 DNA was purified using phenol-chloroform extraction and concentrated by ethanol precipitation. Each chromatin extract was then used as an input control and for two separate immunoprecipitations (H3K4me1 and H4K4me3), thus INE1 is the input both for the M1E1 and M3E1, INE2 for the M1E2 and M3E2, INM1 for M1M1 and M3M1 and INM2 for M1M2 and M3M2. After crosslinking, spheroplasts were isolated as described before using zymolase and resuspended in 0.3 ml of lysis buffer (50 mM Hepes-KOH at pH 7.2, 140 mM NaCl, 1 mM EDTA, 0.1% Deoxycholic acid sodium salt and 1% Triton X-100) containing a cocktail of protease inhibitors (EDTA-free Tablets, Roche). An equal volume of glass beads (0.5-mm diameter) was added, and the spheroplasts were broken using a bead-beater (FastPrep-24, Biomedicals). Glass beads were then removed and the lysate was transferred to a Sorenson tubes to digest the chromatin into fragments of 300 nucleotides using the Bioruptor Pico (30 cycles, 30”on/30”off). The whole extract was clarified by centrifugation for 10 min at 5000 × g at 4 °C and an aliquot was taken as input. In parallel, 50 µl of Dynabeads M-280 Sheep anti Rabbit IgG (Thermo Fisher) per sample were washed twice with PBS+5mg/ml of BSA and incubated overnight at 4ºC with 2.5 µg of the primary antibody (H3K4me1 (ab8895, Abcam) or H3K4me3 (ab8580, Abcam)). Beads were then washed with PBS+5mg/ml of BSA and resuspended in 30 µl/sample of PBS-BSA 5mg/ml. Extracts were then incubated 2h at 4ºC with the Dynabeads, previously conjugated with the primary antibody, and then washed two times with lysis buffer, two times with the lysis buffer supplemented with 360mM NaCl,, 2 times with washing buffer (0.5% Deoxycholic acid sodium salt, 10mM TRIS pH8, 250 mM LiCl, 0.5% NP-40, 1 mM EDTA) and one time with TE (10Mm Tris-HCl pH 7.5,1mM EDTA). Dynabeads were resuspended in 80 µl of Elution Buffer (50 mM TRIS, 10 mM of EDTA, 1% SDS) and crosslinking was reverted incubating overnight at 65ºC followed by 2h incubation at 37ºC with 0.80 mg/ml per sample of proteinase K. ENA first public 2021-03-10 ENA last update 2021-01-12 External Id SAMEA7804230 INSDC center alias Institute for Research in Biomedicine (IRB Barcelona)-The Barcelona Institute of Science and Technology. INSDC center name Institute for Research in Biomedicine (IRB Barcelona)-The Barcelona Institute of Science and Technology. INSDC first public 2021-03-10T04:08:23Z INSDC last update 2021-01-12T20:09:29Z INSDC status public Submitter Id E-MTAB-10001:INM2 broker name ArrayExpress common name baker's yeast genetic modification vector expressing DNMT1, 3a, 3b, 3L genotype Mata ura3-52 lys2-801 ade2-101 trp1-63 his3-200 leu2-1 growth condition Raffinose/Galactose phenotype DNA methylation sample name E-MTAB-10001:INM2 stimulus doxycycline induction strain YPH499 ERS5551484 SAMEA7804231 4932 Saccharomyces cerevisiae Protocols: Yeast cells were collected by centrifugation 10min at 1000x g, washed twice with sterilized water, and resuspended into selective media Strain YPH499 (Mata ura3-52 lys2-801 ade2-101 trp1-Δ63 his3-Δ200 leu2-Δ1) was transformed with 2, 3 or 4 expression plasmids by the standard lithium acetate procedure. Transformants were selected on plates of appropriate selective medium with 2% Raffinose and 10µg/ml doxycycline to repress any expression. Selected transformants (2 to 4 transformants per combination of plasmids) were grown on selective liquid medium with 2% Raffinose and 10µg/ml doxycycline up to OD600=0.5. Then, yeast cells were spun 10min at 1000x g, washed twice with sterilized water, and resuspended into selective media with 1% Raffinose and 2% Galactose without doxycycline to allow expression of all four DNMTs. For experiments on synchronized cells, cells were treated with alpha-factor (3µM final) for 2 to 4 hours to synchronise cells in G1 DNA was purified using phenol-chloroform extraction and concentrated by ethanol precipitation. Each chromatin extract was then used as an input control and for two separate immunoprecipitations (H3K4me1 and H4K4me3), thus INE1 is the input both for the M1E1 and M3E1, INE2 for the M1E2 and M3E2, INM1 for M1M1 and M3M1 and INM2 for M1M2 and M3M2. After crosslinking, spheroplasts were isolated as described before using zymolase and resuspended in 0.3 ml of lysis buffer (50 mM Hepes-KOH at pH 7.2, 140 mM NaCl, 1 mM EDTA, 0.1% Deoxycholic acid sodium salt and 1% Triton X-100) containing a cocktail of protease inhibitors (EDTA-free Tablets, Roche). An equal volume of glass beads (0.5-mm diameter) was added, and the spheroplasts were broken using a bead-beater (FastPrep-24, Biomedicals). Glass beads were then removed and the lysate was transferred to a Sorenson tubes to digest the chromatin into fragments of 300 nucleotides using the Bioruptor Pico (30 cycles, 30”on/30”off). The whole extract was clarified by centrifugation for 10 min at 5000 × g at 4 °C and an aliquot was taken as input. In parallel, 50 µl of Dynabeads M-280 Sheep anti Rabbit IgG (Thermo Fisher) per sample were washed twice with PBS+5mg/ml of BSA and incubated overnight at 4ºC with 2.5 µg of the primary antibody (H3K4me1 (ab8895, Abcam) or H3K4me3 (ab8580, Abcam)). Beads were then washed with PBS+5mg/ml of BSA and resuspended in 30 µl/sample of PBS-BSA 5mg/ml. Extracts were then incubated 2h at 4ºC with the Dynabeads, previously conjugated with the primary antibody, and then washed two times with lysis buffer, two times with the lysis buffer supplemented with 360mM NaCl,, 2 times with washing buffer (0.5% Deoxycholic acid sodium salt, 10mM TRIS pH8, 250 mM LiCl, 0.5% NP-40, 1 mM EDTA) and one time with TE (10Mm Tris-HCl pH 7.5,1mM EDTA). Dynabeads were resuspended in 80 µl of Elution Buffer (50 mM TRIS, 10 mM of EDTA, 1% SDS) and crosslinking was reverted incubating overnight at 65ºC followed by 2h incubation at 37ºC with 0.80 mg/ml per sample of proteinase K. ENA first public 2021-03-10 ENA last update 2021-01-12 External Id SAMEA7804231 INSDC center alias Institute for Research in Biomedicine (IRB Barcelona)-The Barcelona Institute of Science and Technology. INSDC center name Institute for Research in Biomedicine (IRB Barcelona)-The Barcelona Institute of Science and Technology. INSDC first public 2021-03-10T04:08:23Z INSDC last update 2021-01-12T20:09:29Z INSDC status public Submitter Id E-MTAB-10001:M1E1 broker name ArrayExpress common name baker's yeast genetic modification empty vector genotype Mata ura3-52 lys2-801 ade2-101 trp1-63 his3-200 leu2-1 growth condition Raffinose/Galactose phenotype wild type sample name E-MTAB-10001:M1E1 strain YPH499 ERS5551485 SAMEA7804232 4932 Saccharomyces cerevisiae Protocols: Yeast cells were collected by centrifugation 10min at 1000x g, washed twice with sterilized water, and resuspended into selective media Strain YPH499 (Mata ura3-52 lys2-801 ade2-101 trp1-Δ63 his3-Δ200 leu2-Δ1) was transformed with 2, 3 or 4 expression plasmids by the standard lithium acetate procedure. Transformants were selected on plates of appropriate selective medium with 2% Raffinose and 10µg/ml doxycycline to repress any expression. Selected transformants (2 to 4 transformants per combination of plasmids) were grown on selective liquid medium with 2% Raffinose and 10µg/ml doxycycline up to OD600=0.5. Then, yeast cells were spun 10min at 1000x g, washed twice with sterilized water, and resuspended into selective media with 1% Raffinose and 2% Galactose without doxycycline to allow expression of all four DNMTs. For experiments on synchronized cells, cells were treated with alpha-factor (3µM final) for 2 to 4 hours to synchronise cells in G1 DNA was purified using phenol-chloroform extraction and concentrated by ethanol precipitation. Each chromatin extract was then used as an input control and for two separate immunoprecipitations (H3K4me1 and H4K4me3), thus INE1 is the input both for the M1E1 and M3E1, INE2 for the M1E2 and M3E2, INM1 for M1M1 and M3M1 and INM2 for M1M2 and M3M2. After crosslinking, spheroplasts were isolated as described before using zymolase and resuspended in 0.3 ml of lysis buffer (50 mM Hepes-KOH at pH 7.2, 140 mM NaCl, 1 mM EDTA, 0.1% Deoxycholic acid sodium salt and 1% Triton X-100) containing a cocktail of protease inhibitors (EDTA-free Tablets, Roche). An equal volume of glass beads (0.5-mm diameter) was added, and the spheroplasts were broken using a bead-beater (FastPrep-24, Biomedicals). Glass beads were then removed and the lysate was transferred to a Sorenson tubes to digest the chromatin into fragments of 300 nucleotides using the Bioruptor Pico (30 cycles, 30”on/30”off). The whole extract was clarified by centrifugation for 10 min at 5000 × g at 4 °C and an aliquot was taken as input. In parallel, 50 µl of Dynabeads M-280 Sheep anti Rabbit IgG (Thermo Fisher) per sample were washed twice with PBS+5mg/ml of BSA and incubated overnight at 4ºC with 2.5 µg of the primary antibody (H3K4me1 (ab8895, Abcam) or H3K4me3 (ab8580, Abcam)). Beads were then washed with PBS+5mg/ml of BSA and resuspended in 30 µl/sample of PBS-BSA 5mg/ml. Extracts were then incubated 2h at 4ºC with the Dynabeads, previously conjugated with the primary antibody, and then washed two times with lysis buffer, two times with the lysis buffer supplemented with 360mM NaCl,, 2 times with washing buffer (0.5% Deoxycholic acid sodium salt, 10mM TRIS pH8, 250 mM LiCl, 0.5% NP-40, 1 mM EDTA) and one time with TE (10Mm Tris-HCl pH 7.5,1mM EDTA). Dynabeads were resuspended in 80 µl of Elution Buffer (50 mM TRIS, 10 mM of EDTA, 1% SDS) and crosslinking was reverted incubating overnight at 65ºC followed by 2h incubation at 37ºC with 0.80 mg/ml per sample of proteinase K. ENA first public 2021-03-10 ENA last update 2021-01-12 External Id SAMEA7804232 INSDC center alias Institute for Research in Biomedicine (IRB Barcelona)-The Barcelona Institute of Science and Technology. INSDC center name Institute for Research in Biomedicine (IRB Barcelona)-The Barcelona Institute of Science and Technology. INSDC first public 2021-03-10T04:08:23Z INSDC last update 2021-01-12T20:09:29Z INSDC status public Submitter Id E-MTAB-10001:M1E2 broker name ArrayExpress common name baker's yeast genetic modification empty vector genotype Mata ura3-52 lys2-801 ade2-101 trp1-63 his3-200 leu2-1 growth condition Raffinose/Galactose phenotype wild type sample name E-MTAB-10001:M1E2 strain YPH499 ERS5551487 SAMEA7804234 4932 Saccharomyces cerevisiae Protocols: Yeast cells were collected by centrifugation 10min at 1000x g, washed twice with sterilized water, and resuspended into selective media Strain YPH499 (Mata ura3-52 lys2-801 ade2-101 trp1-Δ63 his3-Δ200 leu2-Δ1) was transformed with 2, 3 or 4 expression plasmids by the standard lithium acetate procedure. Transformants were selected on plates of appropriate selective medium with 2% Raffinose and 10µg/ml doxycycline to repress any expression. Selected transformants (2 to 4 transformants per combination of plasmids) were grown on selective liquid medium with 2% Raffinose and 10µg/ml doxycycline up to OD600=0.5. Then, yeast cells were spun 10min at 1000x g, washed twice with sterilized water, and resuspended into selective media with 1% Raffinose and 2% Galactose without doxycycline to allow expression of all four DNMTs. For experiments on synchronized cells, cells were treated with alpha-factor (3µM final) for 2 to 4 hours to synchronise cells in G1 DNA was purified using phenol-chloroform extraction and concentrated by ethanol precipitation. Each chromatin extract was then used as an input control and for two separate immunoprecipitations (H3K4me1 and H4K4me3), thus INE1 is the input both for the M1E1 and M3E1, INE2 for the M1E2 and M3E2, INM1 for M1M1 and M3M1 and INM2 for M1M2 and M3M2. After crosslinking, spheroplasts were isolated as described before using zymolase and resuspended in 0.3 ml of lysis buffer (50 mM Hepes-KOH at pH 7.2, 140 mM NaCl, 1 mM EDTA, 0.1% Deoxycholic acid sodium salt and 1% Triton X-100) containing a cocktail of protease inhibitors (EDTA-free Tablets, Roche). An equal volume of glass beads (0.5-mm diameter) was added, and the spheroplasts were broken using a bead-beater (FastPrep-24, Biomedicals). Glass beads were then removed and the lysate was transferred to a Sorenson tubes to digest the chromatin into fragments of 300 nucleotides using the Bioruptor Pico (30 cycles, 30”on/30”off). The whole extract was clarified by centrifugation for 10 min at 5000 × g at 4 °C and an aliquot was taken as input. In parallel, 50 µl of Dynabeads M-280 Sheep anti Rabbit IgG (Thermo Fisher) per sample were washed twice with PBS+5mg/ml of BSA and incubated overnight at 4ºC with 2.5 µg of the primary antibody (H3K4me1 (ab8895, Abcam) or H3K4me3 (ab8580, Abcam)). Beads were then washed with PBS+5mg/ml of BSA and resuspended in 30 µl/sample of PBS-BSA 5mg/ml. Extracts were then incubated 2h at 4ºC with the Dynabeads, previously conjugated with the primary antibody, and then washed two times with lysis buffer, two times with the lysis buffer supplemented with 360mM NaCl,, 2 times with washing buffer (0.5% Deoxycholic acid sodium salt, 10mM TRIS pH8, 250 mM LiCl, 0.5% NP-40, 1 mM EDTA) and one time with TE (10Mm Tris-HCl pH 7.5,1mM EDTA). Dynabeads were resuspended in 80 µl of Elution Buffer (50 mM TRIS, 10 mM of EDTA, 1% SDS) and crosslinking was reverted incubating overnight at 65ºC followed by 2h incubation at 37ºC with 0.80 mg/ml per sample of proteinase K. ENA first public 2021-03-10 ENA last update 2021-01-12 External Id SAMEA7804234 INSDC center alias Institute for Research in Biomedicine (IRB Barcelona)-The Barcelona Institute of Science and Technology. INSDC center name Institute for Research in Biomedicine (IRB Barcelona)-The Barcelona Institute of Science and Technology. INSDC first public 2021-03-10T04:08:23Z INSDC last update 2021-01-12T20:09:29Z INSDC status public Submitter Id E-MTAB-10001:M1M2 broker name ArrayExpress common name baker's yeast genetic modification vector expressing DNMT1, 3a, 3b, 3L genotype Mata ura3-52 lys2-801 ade2-101 trp1-63 his3-200 leu2-1 growth condition Raffinose/Galactose phenotype DNA methylation sample name E-MTAB-10001:M1M2 stimulus doxycycline induction strain YPH499 ERS5551488 SAMEA7804235 4932 Saccharomyces cerevisiae Protocols: Yeast cells were collected by centrifugation 10min at 1000x g, washed twice with sterilized water, and resuspended into selective media Strain YPH499 (Mata ura3-52 lys2-801 ade2-101 trp1-Δ63 his3-Δ200 leu2-Δ1) was transformed with 2, 3 or 4 expression plasmids by the standard lithium acetate procedure. Transformants were selected on plates of appropriate selective medium with 2% Raffinose and 10µg/ml doxycycline to repress any expression. Selected transformants (2 to 4 transformants per combination of plasmids) were grown on selective liquid medium with 2% Raffinose and 10µg/ml doxycycline up to OD600=0.5. Then, yeast cells were spun 10min at 1000x g, washed twice with sterilized water, and resuspended into selective media with 1% Raffinose and 2% Galactose without doxycycline to allow expression of all four DNMTs. For experiments on synchronized cells, cells were treated with alpha-factor (3µM final) for 2 to 4 hours to synchronise cells in G1 DNA was purified using phenol-chloroform extraction and concentrated by ethanol precipitation. Each chromatin extract was then used as an input control and for two separate immunoprecipitations (H3K4me1 and H4K4me3), thus INE1 is the input both for the M1E1 and M3E1, INE2 for the M1E2 and M3E2, INM1 for M1M1 and M3M1 and INM2 for M1M2 and M3M2. After crosslinking, spheroplasts were isolated as described before using zymolase and resuspended in 0.3 ml of lysis buffer (50 mM Hepes-KOH at pH 7.2, 140 mM NaCl, 1 mM EDTA, 0.1% Deoxycholic acid sodium salt and 1% Triton X-100) containing a cocktail of protease inhibitors (EDTA-free Tablets, Roche). An equal volume of glass beads (0.5-mm diameter) was added, and the spheroplasts were broken using a bead-beater (FastPrep-24, Biomedicals). Glass beads were then removed and the lysate was transferred to a Sorenson tubes to digest the chromatin into fragments of 300 nucleotides using the Bioruptor Pico (30 cycles, 30”on/30”off). The whole extract was clarified by centrifugation for 10 min at 5000 × g at 4 °C and an aliquot was taken as input. In parallel, 50 µl of Dynabeads M-280 Sheep anti Rabbit IgG (Thermo Fisher) per sample were washed twice with PBS+5mg/ml of BSA and incubated overnight at 4ºC with 2.5 µg of the primary antibody (H3K4me1 (ab8895, Abcam) or H3K4me3 (ab8580, Abcam)). Beads were then washed with PBS+5mg/ml of BSA and resuspended in 30 µl/sample of PBS-BSA 5mg/ml. Extracts were then incubated 2h at 4ºC with the Dynabeads, previously conjugated with the primary antibody, and then washed two times with lysis buffer, two times with the lysis buffer supplemented with 360mM NaCl,, 2 times with washing buffer (0.5% Deoxycholic acid sodium salt, 10mM TRIS pH8, 250 mM LiCl, 0.5% NP-40, 1 mM EDTA) and one time with TE (10Mm Tris-HCl pH 7.5,1mM EDTA). Dynabeads were resuspended in 80 µl of Elution Buffer (50 mM TRIS, 10 mM of EDTA, 1% SDS) and crosslinking was reverted incubating overnight at 65ºC followed by 2h incubation at 37ºC with 0.80 mg/ml per sample of proteinase K. ENA first public 2021-03-10 ENA last update 2021-01-12 External Id SAMEA7804235 INSDC center alias Institute for Research in Biomedicine (IRB Barcelona)-The Barcelona Institute of Science and Technology. INSDC center name Institute for Research in Biomedicine (IRB Barcelona)-The Barcelona Institute of Science and Technology. INSDC first public 2021-03-10T04:08:23Z INSDC last update 2021-01-12T20:09:29Z INSDC status public Submitter Id E-MTAB-10001:M3E1 broker name ArrayExpress common name baker's yeast genetic modification empty vector genotype Mata ura3-52 lys2-801 ade2-101 trp1-63 his3-200 leu2-1 growth condition Raffinose/Galactose phenotype wild type sample name E-MTAB-10001:M3E1 strain YPH499 ERS5551489 SAMEA7804236 4932 Saccharomyces cerevisiae Protocols: Yeast cells were collected by centrifugation 10min at 1000x g, washed twice with sterilized water, and resuspended into selective media Strain YPH499 (Mata ura3-52 lys2-801 ade2-101 trp1-Δ63 his3-Δ200 leu2-Δ1) was transformed with 2, 3 or 4 expression plasmids by the standard lithium acetate procedure. Transformants were selected on plates of appropriate selective medium with 2% Raffinose and 10µg/ml doxycycline to repress any expression. Selected transformants (2 to 4 transformants per combination of plasmids) were grown on selective liquid medium with 2% Raffinose and 10µg/ml doxycycline up to OD600=0.5. Then, yeast cells were spun 10min at 1000x g, washed twice with sterilized water, and resuspended into selective media with 1% Raffinose and 2% Galactose without doxycycline to allow expression of all four DNMTs. For experiments on synchronized cells, cells were treated with alpha-factor (3µM final) for 2 to 4 hours to synchronise cells in G1 DNA was purified using phenol-chloroform extraction and concentrated by ethanol precipitation. Each chromatin extract was then used as an input control and for two separate immunoprecipitations (H3K4me1 and H4K4me3), thus INE1 is the input both for the M1E1 and M3E1, INE2 for the M1E2 and M3E2, INM1 for M1M1 and M3M1 and INM2 for M1M2 and M3M2. After crosslinking, spheroplasts were isolated as described before using zymolase and resuspended in 0.3 ml of lysis buffer (50 mM Hepes-KOH at pH 7.2, 140 mM NaCl, 1 mM EDTA, 0.1% Deoxycholic acid sodium salt and 1% Triton X-100) containing a cocktail of protease inhibitors (EDTA-free Tablets, Roche). An equal volume of glass beads (0.5-mm diameter) was added, and the spheroplasts were broken using a bead-beater (FastPrep-24, Biomedicals). Glass beads were then removed and the lysate was transferred to a Sorenson tubes to digest the chromatin into fragments of 300 nucleotides using the Bioruptor Pico (30 cycles, 30”on/30”off). The whole extract was clarified by centrifugation for 10 min at 5000 × g at 4 °C and an aliquot was taken as input. In parallel, 50 µl of Dynabeads M-280 Sheep anti Rabbit IgG (Thermo Fisher) per sample were washed twice with PBS+5mg/ml of BSA and incubated overnight at 4ºC with 2.5 µg of the primary antibody (H3K4me1 (ab8895, Abcam) or H3K4me3 (ab8580, Abcam)). Beads were then washed with PBS+5mg/ml of BSA and resuspended in 30 µl/sample of PBS-BSA 5mg/ml. Extracts were then incubated 2h at 4ºC with the Dynabeads, previously conjugated with the primary antibody, and then washed two times with lysis buffer, two times with the lysis buffer supplemented with 360mM NaCl,, 2 times with washing buffer (0.5% Deoxycholic acid sodium salt, 10mM TRIS pH8, 250 mM LiCl, 0.5% NP-40, 1 mM EDTA) and one time with TE (10Mm Tris-HCl pH 7.5,1mM EDTA). Dynabeads were resuspended in 80 µl of Elution Buffer (50 mM TRIS, 10 mM of EDTA, 1% SDS) and crosslinking was reverted incubating overnight at 65ºC followed by 2h incubation at 37ºC with 0.80 mg/ml per sample of proteinase K. ENA first public 2021-03-10 ENA last update 2021-01-12 External Id SAMEA7804236 INSDC center alias Institute for Research in Biomedicine (IRB Barcelona)-The Barcelona Institute of Science and Technology. INSDC center name Institute for Research in Biomedicine (IRB Barcelona)-The Barcelona Institute of Science and Technology. INSDC first public 2021-03-10T04:08:23Z INSDC last update 2021-01-12T20:09:30Z INSDC status public Submitter Id E-MTAB-10001:M3E2 broker name ArrayExpress common name baker's yeast genetic modification empty vector genotype Mata ura3-52 lys2-801 ade2-101 trp1-63 his3-200 leu2-1 growth condition Raffinose/Galactose phenotype wild type sample name E-MTAB-10001:M3E2 strain YPH499 ERS5551490 SAMEA7804237 4932 Saccharomyces cerevisiae Protocols: Yeast cells were collected by centrifugation 10min at 1000x g, washed twice with sterilized water, and resuspended into selective media Strain YPH499 (Mata ura3-52 lys2-801 ade2-101 trp1-Δ63 his3-Δ200 leu2-Δ1) was transformed with 2, 3 or 4 expression plasmids by the standard lithium acetate procedure. Transformants were selected on plates of appropriate selective medium with 2% Raffinose and 10µg/ml doxycycline to repress any expression. Selected transformants (2 to 4 transformants per combination of plasmids) were grown on selective liquid medium with 2% Raffinose and 10µg/ml doxycycline up to OD600=0.5. Then, yeast cells were spun 10min at 1000x g, washed twice with sterilized water, and resuspended into selective media with 1% Raffinose and 2% Galactose without doxycycline to allow expression of all four DNMTs. For experiments on synchronized cells, cells were treated with alpha-factor (3µM final) for 2 to 4 hours to synchronise cells in G1 DNA was purified using phenol-chloroform extraction and concentrated by ethanol precipitation. Each chromatin extract was then used as an input control and for two separate immunoprecipitations (H3K4me1 and H4K4me3), thus INE1 is the input both for the M1E1 and M3E1, INE2 for the M1E2 and M3E2, INM1 for M1M1 and M3M1 and INM2 for M1M2 and M3M2. After crosslinking, spheroplasts were isolated as described before using zymolase and resuspended in 0.3 ml of lysis buffer (50 mM Hepes-KOH at pH 7.2, 140 mM NaCl, 1 mM EDTA, 0.1% Deoxycholic acid sodium salt and 1% Triton X-100) containing a cocktail of protease inhibitors (EDTA-free Tablets, Roche). An equal volume of glass beads (0.5-mm diameter) was added, and the spheroplasts were broken using a bead-beater (FastPrep-24, Biomedicals). Glass beads were then removed and the lysate was transferred to a Sorenson tubes to digest the chromatin into fragments of 300 nucleotides using the Bioruptor Pico (30 cycles, 30”on/30”off). The whole extract was clarified by centrifugation for 10 min at 5000 × g at 4 °C and an aliquot was taken as input. In parallel, 50 µl of Dynabeads M-280 Sheep anti Rabbit IgG (Thermo Fisher) per sample were washed twice with PBS+5mg/ml of BSA and incubated overnight at 4ºC with 2.5 µg of the primary antibody (H3K4me1 (ab8895, Abcam) or H3K4me3 (ab8580, Abcam)). Beads were then washed with PBS+5mg/ml of BSA and resuspended in 30 µl/sample of PBS-BSA 5mg/ml. Extracts were then incubated 2h at 4ºC with the Dynabeads, previously conjugated with the primary antibody, and then washed two times with lysis buffer, two times with the lysis buffer supplemented with 360mM NaCl,, 2 times with washing buffer (0.5% Deoxycholic acid sodium salt, 10mM TRIS pH8, 250 mM LiCl, 0.5% NP-40, 1 mM EDTA) and one time with TE (10Mm Tris-HCl pH 7.5,1mM EDTA). Dynabeads were resuspended in 80 µl of Elution Buffer (50 mM TRIS, 10 mM of EDTA, 1% SDS) and crosslinking was reverted incubating overnight at 65ºC followed by 2h incubation at 37ºC with 0.80 mg/ml per sample of proteinase K. ENA first public 2021-03-10 ENA last update 2021-01-12 External Id SAMEA7804237 INSDC center alias Institute for Research in Biomedicine (IRB Barcelona)-The Barcelona Institute of Science and Technology. INSDC center name Institute for Research in Biomedicine (IRB Barcelona)-The Barcelona Institute of Science and Technology. INSDC first public 2021-03-10T04:08:23Z INSDC last update 2021-01-12T20:09:30Z INSDC status public Submitter Id E-MTAB-10001:M3M1 broker name ArrayExpress common name baker's yeast genetic modification vector expressing DNMT1, 3a, 3b, 3L genotype Mata ura3-52 lys2-801 ade2-101 trp1-63 his3-200 leu2-1 growth condition Raffinose/Galactose phenotype DNA methylation sample name E-MTAB-10001:M3M1 stimulus doxycycline induction strain YPH499 ERS5551491 SAMEA7804238 4932 Saccharomyces cerevisiae Protocols: Yeast cells were collected by centrifugation 10min at 1000x g, washed twice with sterilized water, and resuspended into selective media Strain YPH499 (Mata ura3-52 lys2-801 ade2-101 trp1-Δ63 his3-Δ200 leu2-Δ1) was transformed with 2, 3 or 4 expression plasmids by the standard lithium acetate procedure. Transformants were selected on plates of appropriate selective medium with 2% Raffinose and 10µg/ml doxycycline to repress any expression. Selected transformants (2 to 4 transformants per combination of plasmids) were grown on selective liquid medium with 2% Raffinose and 10µg/ml doxycycline up to OD600=0.5. Then, yeast cells were spun 10min at 1000x g, washed twice with sterilized water, and resuspended into selective media with 1% Raffinose and 2% Galactose without doxycycline to allow expression of all four DNMTs. For experiments on synchronized cells, cells were treated with alpha-factor (3µM final) for 2 to 4 hours to synchronise cells in G1 DNA was purified using phenol-chloroform extraction and concentrated by ethanol precipitation. Each chromatin extract was then used as an input control and for two separate immunoprecipitations (H3K4me1 and H4K4me3), thus INE1 is the input both for the M1E1 and M3E1, INE2 for the M1E2 and M3E2, INM1 for M1M1 and M3M1 and INM2 for M1M2 and M3M2. After crosslinking, spheroplasts were isolated as described before using zymolase and resuspended in 0.3 ml of lysis buffer (50 mM Hepes-KOH at pH 7.2, 140 mM NaCl, 1 mM EDTA, 0.1% Deoxycholic acid sodium salt and 1% Triton X-100) containing a cocktail of protease inhibitors (EDTA-free Tablets, Roche). An equal volume of glass beads (0.5-mm diameter) was added, and the spheroplasts were broken using a bead-beater (FastPrep-24, Biomedicals). Glass beads were then removed and the lysate was transferred to a Sorenson tubes to digest the chromatin into fragments of 300 nucleotides using the Bioruptor Pico (30 cycles, 30”on/30”off). The whole extract was clarified by centrifugation for 10 min at 5000 × g at 4 °C and an aliquot was taken as input. In parallel, 50 µl of Dynabeads M-280 Sheep anti Rabbit IgG (Thermo Fisher) per sample were washed twice with PBS+5mg/ml of BSA and incubated overnight at 4ºC with 2.5 µg of the primary antibody (H3K4me1 (ab8895, Abcam) or H3K4me3 (ab8580, Abcam)). Beads were then washed with PBS+5mg/ml of BSA and resuspended in 30 µl/sample of PBS-BSA 5mg/ml. Extracts were then incubated 2h at 4ºC with the Dynabeads, previously conjugated with the primary antibody, and then washed two times with lysis buffer, two times with the lysis buffer supplemented with 360mM NaCl,, 2 times with washing buffer (0.5% Deoxycholic acid sodium salt, 10mM TRIS pH8, 250 mM LiCl, 0.5% NP-40, 1 mM EDTA) and one time with TE (10Mm Tris-HCl pH 7.5,1mM EDTA). Dynabeads were resuspended in 80 µl of Elution Buffer (50 mM TRIS, 10 mM of EDTA, 1% SDS) and crosslinking was reverted incubating overnight at 65ºC followed by 2h incubation at 37ºC with 0.80 mg/ml per sample of proteinase K. ENA first public 2021-03-10 ENA last update 2021-01-12 External Id SAMEA7804238 INSDC center alias Institute for Research in Biomedicine (IRB Barcelona)-The Barcelona Institute of Science and Technology. INSDC center name Institute for Research in Biomedicine (IRB Barcelona)-The Barcelona Institute of Science and Technology. INSDC first public 2021-03-10T04:08:23Z INSDC last update 2021-01-12T20:09:30Z INSDC status public Submitter Id E-MTAB-10001:M3M2 broker name ArrayExpress common name baker's yeast genetic modification vector expressing DNMT1, 3a, 3b, 3L genotype Mata ura3-52 lys2-801 ade2-101 trp1-63 his3-200 leu2-1 growth condition Raffinose/Galactose phenotype DNA methylation sample name E-MTAB-10001:M3M2 stimulus doxycycline induction strain YPH499 ERS5551486 SAMEA7804233 4932 Saccharomyces cerevisiae Protocols: Yeast cells were collected by centrifugation 10min at 1000x g, washed twice with sterilized water, and resuspended into selective media Strain YPH499 (Mata ura3-52 lys2-801 ade2-101 trp1-Δ63 his3-Δ200 leu2-Δ1) was transformed with 2, 3 or 4 expression plasmids by the standard lithium acetate procedure. Transformants were selected on plates of appropriate selective medium with 2% Raffinose and 10µg/ml doxycycline to repress any expression. Selected transformants (2 to 4 transformants per combination of plasmids) were grown on selective liquid medium with 2% Raffinose and 10µg/ml doxycycline up to OD600=0.5. Then, yeast cells were spun 10min at 1000x g, washed twice with sterilized water, and resuspended into selective media with 1% Raffinose and 2% Galactose without doxycycline to allow expression of all four DNMTs. For experiments on synchronized cells, cells were treated with alpha-factor (3µM final) for 2 to 4 hours to synchronise cells in G1 DNA was purified using phenol-chloroform extraction and concentrated by ethanol precipitation. Each chromatin extract was then used as an input control and for two separate immunoprecipitations (H3K4me1 and H4K4me3), thus INE1 is the input both for the M1E1 and M3E1, INE2 for the M1E2 and M3E2, INM1 for M1M1 and M3M1 and INM2 for M1M2 and M3M2. After crosslinking, spheroplasts were isolated as described before using zymolase and resuspended in 0.3 ml of lysis buffer (50 mM Hepes-KOH at pH 7.2, 140 mM NaCl, 1 mM EDTA, 0.1% Deoxycholic acid sodium salt and 1% Triton X-100) containing a cocktail of protease inhibitors (EDTA-free Tablets, Roche). An equal volume of glass beads (0.5-mm diameter) was added, and the spheroplasts were broken using a bead-beater (FastPrep-24, Biomedicals). Glass beads were then removed and the lysate was transferred to a Sorenson tubes to digest the chromatin into fragments of 300 nucleotides using the Bioruptor Pico (30 cycles, 30”on/30”off). The whole extract was clarified by centrifugation for 10 min at 5000 × g at 4 °C and an aliquot was taken as input. In parallel, 50 µl of Dynabeads M-280 Sheep anti Rabbit IgG (Thermo Fisher) per sample were washed twice with PBS+5mg/ml of BSA and incubated overnight at 4ºC with 2.5 µg of the primary antibody (H3K4me1 (ab8895, Abcam) or H3K4me3 (ab8580, Abcam)). Beads were then washed with PBS+5mg/ml of BSA and resuspended in 30 µl/sample of PBS-BSA 5mg/ml. Extracts were then incubated 2h at 4ºC with the Dynabeads, previously conjugated with the primary antibody, and then washed two times with lysis buffer, two times with the lysis buffer supplemented with 360mM NaCl,, 2 times with washing buffer (0.5% Deoxycholic acid sodium salt, 10mM TRIS pH8, 250 mM LiCl, 0.5% NP-40, 1 mM EDTA) and one time with TE (10Mm Tris-HCl pH 7.5,1mM EDTA). Dynabeads were resuspended in 80 µl of Elution Buffer (50 mM TRIS, 10 mM of EDTA, 1% SDS) and crosslinking was reverted incubating overnight at 65ºC followed by 2h incubation at 37ºC with 0.80 mg/ml per sample of proteinase K. ENA first public 2021-03-10 ENA last update 2021-01-12 External Id SAMEA7804233 INSDC center alias Institute for Research in Biomedicine (IRB Barcelona)-The Barcelona Institute of Science and Technology. INSDC center name Institute for Research in Biomedicine (IRB Barcelona)-The Barcelona Institute of Science and Technology. INSDC first public 2021-03-10T04:08:23Z INSDC last update 2021-01-12T20:09:29Z INSDC status public Submitter Id E-MTAB-10001:M1M1 broker name ArrayExpress common name baker's yeast genetic modification vector expressing DNMT1, 3a, 3b, 3L genotype Mata ura3-52 lys2-801 ade2-101 trp1-63 his3-200 leu2-1 growth condition Raffinose/Galactose phenotype DNA methylation sample name E-MTAB-10001:M1M1 stimulus doxycycline induction strain YPH499