ERS2897137 SAMEA5085786 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:14Z External Id SAMEA5085786 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:14Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_77 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_77 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897148 SAMEA5085797 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:14Z External Id SAMEA5085797 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:14Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_87 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_87 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897136 SAMEA5085785 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:14Z External Id SAMEA5085785 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:14Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_76 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_76 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897150 SAMEA5085799 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:14Z External Id SAMEA5085799 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:14Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_89 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_89 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897151 SAMEA5085800 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:14Z External Id SAMEA5085800 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:14Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_9 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_9 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897152 SAMEA5085801 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:14Z External Id SAMEA5085801 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:14Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_90 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_90 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897153 SAMEA5085802 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:14Z External Id SAMEA5085802 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:14Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_91 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_91 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897154 SAMEA5085803 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:14Z External Id SAMEA5085803 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:14Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_92 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_92 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897135 SAMEA5085784 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:14Z External Id SAMEA5085784 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:14Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_75 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_75 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897156 SAMEA5085805 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:15Z External Id SAMEA5085805 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:15Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_94 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_94 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897157 SAMEA5085806 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:15Z External Id SAMEA5085806 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:15Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_95 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_95 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897158 SAMEA5085807 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:15Z External Id SAMEA5085807 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:15Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_96 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_96 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897159 SAMEA5085808 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:15Z External Id SAMEA5085808 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:15Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_1 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_1 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897160 SAMEA5085809 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:15Z External Id SAMEA5085809 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:15Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_10 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_10 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897161 SAMEA5085810 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:15Z External Id SAMEA5085810 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:15Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_11 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_11 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897162 SAMEA5085811 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:15Z External Id SAMEA5085811 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:15Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_12 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_12 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897163 SAMEA5085812 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:15Z External Id SAMEA5085812 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:15Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_13 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_13 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897164 SAMEA5085813 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:15Z External Id SAMEA5085813 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:15Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_14 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_14 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897134 SAMEA5085783 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:14Z External Id SAMEA5085783 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:14Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_74 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_74 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897166 SAMEA5085815 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:15Z External Id SAMEA5085815 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:15Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_16 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_16 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897167 SAMEA5085816 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:15Z External Id SAMEA5085816 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:15Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_17 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_17 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897168 SAMEA5085817 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:15Z External Id SAMEA5085817 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:15Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_18 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_18 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897169 SAMEA5085818 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:15Z External Id SAMEA5085818 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:15Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_19 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_19 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897170 SAMEA5085819 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:15Z External Id SAMEA5085819 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:15Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_2 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_2 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897133 SAMEA5085782 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:14Z External Id SAMEA5085782 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:14Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_73 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_73 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897172 SAMEA5085821 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:15Z External Id SAMEA5085821 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:15Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_21 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_21 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897173 SAMEA5085822 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:15Z External Id SAMEA5085822 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:15Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_22 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_22 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897174 SAMEA5085823 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:15Z External Id SAMEA5085823 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:15Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_23 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_23 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897175 SAMEA5085824 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:15Z External Id SAMEA5085824 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:15Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_24 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_24 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897176 SAMEA5085825 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:15Z External Id SAMEA5085825 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:15Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_25 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_25 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897177 SAMEA5085826 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:15Z External Id SAMEA5085826 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:15Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_26 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_26 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897178 SAMEA5085827 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:15Z External Id SAMEA5085827 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:15Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_27 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_27 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897179 SAMEA5085828 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:15Z External Id SAMEA5085828 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:15Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_28 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_28 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897180 SAMEA5085829 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:15Z External Id SAMEA5085829 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:15Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_29 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_29 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897181 SAMEA5085830 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:15Z External Id SAMEA5085830 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:15Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_3 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_3 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897132 SAMEA5085781 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:14Z External Id SAMEA5085781 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:14Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_72 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_72 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897183 SAMEA5085832 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:15Z External Id SAMEA5085832 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:15Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_31 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_31 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897184 SAMEA5085833 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:15Z External Id SAMEA5085833 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:15Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_32 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_32 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897123 SAMEA5085772 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:13Z External Id SAMEA5085772 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:13Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_64 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_64 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897124 SAMEA5085773 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:13Z External Id SAMEA5085773 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:13Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_65 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_65 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897125 SAMEA5085774 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:13Z External Id SAMEA5085774 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:13Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_66 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_66 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897126 SAMEA5085775 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:13Z External Id SAMEA5085775 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:13Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_67 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_67 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897127 SAMEA5085776 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:14Z External Id SAMEA5085776 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:14Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_68 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_68 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897128 SAMEA5085777 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:14Z External Id SAMEA5085777 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:14Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_69 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_69 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897129 SAMEA5085778 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:14Z External Id SAMEA5085778 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:14Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_7 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_7 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897130 SAMEA5085779 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:14Z External Id SAMEA5085779 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:14Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_70 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_70 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897131 SAMEA5085780 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:14Z External Id SAMEA5085780 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:14Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_71 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_71 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897185 SAMEA5085834 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:16Z External Id SAMEA5085834 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:16Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_33 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_33 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897186 SAMEA5085835 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:16Z External Id SAMEA5085835 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:16Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_34 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_34 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897187 SAMEA5085836 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:16Z External Id SAMEA5085836 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:16Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_35 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_35 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897189 SAMEA5085838 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:16Z External Id SAMEA5085838 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:16Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_37 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_37 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897190 SAMEA5085839 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:16Z External Id SAMEA5085839 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:16Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_38 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_38 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897191 SAMEA5085840 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:16Z External Id SAMEA5085840 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:16Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_39 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_39 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897192 SAMEA5085841 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:16Z External Id SAMEA5085841 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:16Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_4 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_4 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897193 SAMEA5085842 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:16Z External Id SAMEA5085842 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:16Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_40 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_40 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897194 SAMEA5085843 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:16Z External Id SAMEA5085843 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:16Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_41 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_41 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897195 SAMEA5085844 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:16Z External Id SAMEA5085844 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:16Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_42 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_42 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897196 SAMEA5085845 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:16Z External Id SAMEA5085845 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:16Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_43 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_43 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897197 SAMEA5085846 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:16Z External Id SAMEA5085846 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:16Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_44 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_44 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897199 SAMEA5085848 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:16Z External Id SAMEA5085848 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:16Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_46 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_46 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897200 SAMEA5085849 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:16Z External Id SAMEA5085849 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:16Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_47 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_47 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897201 SAMEA5085850 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:16Z External Id SAMEA5085850 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:16Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_48 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_48 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897202 SAMEA5085851 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:16Z External Id SAMEA5085851 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:16Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_49 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_49 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897203 SAMEA5085852 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:16Z External Id SAMEA5085852 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:16Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_5 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_5 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897205 SAMEA5085854 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:16Z External Id SAMEA5085854 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:16Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_51 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_51 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897206 SAMEA5085855 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:16Z External Id SAMEA5085855 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:16Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_52 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_52 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897207 SAMEA5085856 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:16Z External Id SAMEA5085856 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:16Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_53 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_53 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897208 SAMEA5085857 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:16Z External Id SAMEA5085857 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:16Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_54 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_54 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897209 SAMEA5085858 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:16Z External Id SAMEA5085858 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:16Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_55 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_55 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897210 SAMEA5085859 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:16Z External Id SAMEA5085859 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:16Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_56 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_56 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897211 SAMEA5085860 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:16Z External Id SAMEA5085860 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:16Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_57 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_57 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897212 SAMEA5085861 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:16Z External Id SAMEA5085861 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:16Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_58 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_58 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897213 SAMEA5085862 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:16Z External Id SAMEA5085862 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:16Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_59 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_59 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897214 SAMEA5085863 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:16Z External Id SAMEA5085863 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:16Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_6 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_6 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897216 SAMEA5085865 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:16Z External Id SAMEA5085865 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:16Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_61 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_61 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897217 SAMEA5085866 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:17Z External Id SAMEA5085866 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:17Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_62 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_62 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897218 SAMEA5085867 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:17Z External Id SAMEA5085867 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:17Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_63 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_63 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897219 SAMEA5085868 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:17Z External Id SAMEA5085868 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:17Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_64 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_64 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897220 SAMEA5085869 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:17Z External Id SAMEA5085869 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:17Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_65 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_65 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897222 SAMEA5085871 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:17Z External Id SAMEA5085871 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:17Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_67 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_67 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897223 SAMEA5085872 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:17Z External Id SAMEA5085872 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:17Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_68 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_68 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897224 SAMEA5085873 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:17Z External Id SAMEA5085873 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:17Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_69 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_69 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897225 SAMEA5085874 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:17Z External Id SAMEA5085874 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:17Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_7 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_7 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897226 SAMEA5085875 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:17Z External Id SAMEA5085875 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:17Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_70 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_70 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897227 SAMEA5085876 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:17Z External Id SAMEA5085876 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:17Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_71 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_71 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897228 SAMEA5085877 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:17Z External Id SAMEA5085877 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:17Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_72 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_72 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897229 SAMEA5085878 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:17Z External Id SAMEA5085878 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:17Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_73 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_73 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897230 SAMEA5085879 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:17Z External Id SAMEA5085879 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:17Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_74 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_74 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897232 SAMEA5085881 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:17Z External Id SAMEA5085881 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:17Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_76 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_76 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897233 SAMEA5085882 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:17Z External Id SAMEA5085882 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:17Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_77 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_77 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897234 SAMEA5085883 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:17Z External Id SAMEA5085883 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:17Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_78 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_78 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897235 SAMEA5085884 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:17Z External Id SAMEA5085884 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:17Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_79 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_79 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897236 SAMEA5085885 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:17Z External Id SAMEA5085885 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:17Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_8 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_8 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897238 SAMEA5085887 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:17Z External Id SAMEA5085887 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:17Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_81 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_81 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897239 SAMEA5085888 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:17Z External Id SAMEA5085888 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:17Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_82 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_82 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897240 SAMEA5085889 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:17Z External Id SAMEA5085889 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:17Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_83 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_83 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897241 SAMEA5085890 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:17Z External Id SAMEA5085890 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:17Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_84 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_84 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897242 SAMEA5085891 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:17Z External Id SAMEA5085891 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:17Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_85 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_85 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897243 SAMEA5085892 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:17Z External Id SAMEA5085892 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:17Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_86 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_86 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897244 SAMEA5085893 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:17Z External Id SAMEA5085893 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:17Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_87 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_87 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897245 SAMEA5085894 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:17Z External Id SAMEA5085894 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:17Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_88 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_88 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897246 SAMEA5085895 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:17Z External Id SAMEA5085895 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:17Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_89 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_89 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897247 SAMEA5085896 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:17Z External Id SAMEA5085896 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:17Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_9 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_9 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897249 SAMEA5085898 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:17Z External Id SAMEA5085898 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:17Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_91 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_91 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897250 SAMEA5085899 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:17Z External Id SAMEA5085899 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:17Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_92 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_92 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897251 SAMEA5085900 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:17Z External Id SAMEA5085900 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:17Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_93 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_93 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897252 SAMEA5085901 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:18Z External Id SAMEA5085901 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:18Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_94 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_94 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897253 SAMEA5085902 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:18Z External Id SAMEA5085902 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:18Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_95 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_95 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897255 SAMEA5085904 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:18Z External Id SAMEA5085904 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:18Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_1 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_1 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897256 SAMEA5085905 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:18Z External Id SAMEA5085905 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:18Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_10 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_10 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897257 SAMEA5085906 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:18Z External Id SAMEA5085906 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:18Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_11 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_11 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897258 SAMEA5085907 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:18Z External Id SAMEA5085907 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:18Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_12 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_12 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897259 SAMEA5085908 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:18Z External Id SAMEA5085908 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:18Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_13 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_13 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897260 SAMEA5085909 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:18Z External Id SAMEA5085909 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:18Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_14 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_14 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897138 SAMEA5085787 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:14Z External Id SAMEA5085787 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:14Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_78 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_78 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897139 SAMEA5085788 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:14Z External Id SAMEA5085788 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:14Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_79 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_79 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897140 SAMEA5085789 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:14Z External Id SAMEA5085789 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:14Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_8 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_8 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897141 SAMEA5085790 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:14Z External Id SAMEA5085790 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:14Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_80 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_80 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897142 SAMEA5085791 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:14Z External Id SAMEA5085791 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:14Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_81 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_81 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897143 SAMEA5085792 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:14Z External Id SAMEA5085792 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:14Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_82 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_82 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897144 SAMEA5085793 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:14Z External Id SAMEA5085793 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:14Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_83 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_83 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897145 SAMEA5085794 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:14Z External Id SAMEA5085794 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:14Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_84 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_84 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897146 SAMEA5085795 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:14Z External Id SAMEA5085795 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:14Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_85 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_85 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897147 SAMEA5085796 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:14Z External Id SAMEA5085796 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:14Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_86 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_86 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897149 SAMEA5085798 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:14Z External Id SAMEA5085798 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:14Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_88 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_88 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897155 SAMEA5085804 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:15Z External Id SAMEA5085804 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:15Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_10_93 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_10_93 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897165 SAMEA5085814 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:15Z External Id SAMEA5085814 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:15Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_15 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_15 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897171 SAMEA5085820 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:15Z External Id SAMEA5085820 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:15Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_20 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_20 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897182 SAMEA5085831 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:15Z External Id SAMEA5085831 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:15Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_30 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_30 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897188 SAMEA5085837 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:16Z External Id SAMEA5085837 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:16Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_36 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_36 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897198 SAMEA5085847 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:16Z External Id SAMEA5085847 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:16Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_45 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_45 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897204 SAMEA5085853 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:16Z External Id SAMEA5085853 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:16Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_50 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_50 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897261 SAMEA5085910 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:18Z External Id SAMEA5085910 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:18Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_15 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_15 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897262 SAMEA5085911 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:18Z External Id SAMEA5085911 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:18Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_16 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_16 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897263 SAMEA5085912 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:18Z External Id SAMEA5085912 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:18Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_17 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_17 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897265 SAMEA5085914 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:18Z External Id SAMEA5085914 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:18Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_19 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_19 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897266 SAMEA5085915 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:18Z External Id SAMEA5085915 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:18Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_2 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_2 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897267 SAMEA5085916 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:18Z External Id SAMEA5085916 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:18Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_20 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_20 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897268 SAMEA5085917 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:18Z External Id SAMEA5085917 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:18Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_21 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_21 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897269 SAMEA5085918 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:18Z External Id SAMEA5085918 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:18Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_22 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_22 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897271 SAMEA5085920 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:18Z External Id SAMEA5085920 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:18Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_24 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_24 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897272 SAMEA5085921 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:18Z External Id SAMEA5085921 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:18Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_25 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_25 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897273 SAMEA5085922 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:18Z External Id SAMEA5085922 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:18Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_26 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_26 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897274 SAMEA5085923 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:18Z External Id SAMEA5085923 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:18Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_27 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_27 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897275 SAMEA5085924 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:18Z External Id SAMEA5085924 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:18Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_28 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_28 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897276 SAMEA5085925 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:18Z External Id SAMEA5085925 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:18Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_29 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_29 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897277 SAMEA5085926 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:18Z External Id SAMEA5085926 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:18Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_3 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_3 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897278 SAMEA5085927 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:18Z External Id SAMEA5085927 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:18Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_30 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_30 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897279 SAMEA5085928 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:18Z External Id SAMEA5085928 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:18Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_31 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_31 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897280 SAMEA5085929 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:18Z External Id SAMEA5085929 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:18Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_32 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_32 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897282 SAMEA5085931 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:18Z External Id SAMEA5085931 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:18Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_34 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_34 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897283 SAMEA5085932 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:18Z External Id SAMEA5085932 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:18Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_35 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_35 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897284 SAMEA5085933 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:18Z External Id SAMEA5085933 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:18Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_36 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_36 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897285 SAMEA5085934 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:18Z External Id SAMEA5085934 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:18Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_37 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_37 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897286 SAMEA5085935 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:18Z External Id SAMEA5085935 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:18Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_38 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_38 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897288 SAMEA5085937 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:19Z External Id SAMEA5085937 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:19Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_4 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_4 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897289 SAMEA5085938 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:19Z External Id SAMEA5085938 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:19Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_40 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_40 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897290 SAMEA5085939 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:19Z External Id SAMEA5085939 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:19Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_41 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_41 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897291 SAMEA5085940 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:19Z External Id SAMEA5085940 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:19Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_42 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_42 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897292 SAMEA5085941 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:19Z External Id SAMEA5085941 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:19Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_43 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_43 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897293 SAMEA5085942 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:19Z External Id SAMEA5085942 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:19Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_44 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_44 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897294 SAMEA5085943 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:19Z External Id SAMEA5085943 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:19Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_45 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_45 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897295 SAMEA5085944 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:19Z External Id SAMEA5085944 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:19Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_46 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_46 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897296 SAMEA5085945 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:19Z External Id SAMEA5085945 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:19Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_47 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_47 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897298 SAMEA5085947 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:19Z External Id SAMEA5085947 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:19Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_49 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_49 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897299 SAMEA5085948 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:19Z External Id SAMEA5085948 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:19Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_5 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_5 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897300 SAMEA5085949 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:19Z External Id SAMEA5085949 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:19Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_50 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_50 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897301 SAMEA5085950 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:19Z External Id SAMEA5085950 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:19Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_51 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_51 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897302 SAMEA5085951 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:19Z External Id SAMEA5085951 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:19Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_52 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_52 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897304 SAMEA5085953 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:19Z External Id SAMEA5085953 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:19Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_54 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_54 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897305 SAMEA5085954 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:19Z External Id SAMEA5085954 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:19Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_55 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_55 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897306 SAMEA5085955 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:19Z External Id SAMEA5085955 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:19Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_56 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_56 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897307 SAMEA5085956 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:19Z External Id SAMEA5085956 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:19Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_57 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_57 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897308 SAMEA5085957 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:19Z External Id SAMEA5085957 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:19Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_58 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_58 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897309 SAMEA5085958 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:19Z External Id SAMEA5085958 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:19Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_59 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_59 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897310 SAMEA5085959 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:19Z External Id SAMEA5085959 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:19Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_6 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_6 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897311 SAMEA5085960 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:19Z External Id SAMEA5085960 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:19Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_60 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_60 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897312 SAMEA5085961 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:19Z External Id SAMEA5085961 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:19Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_61 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_61 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897313 SAMEA5085962 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:19Z External Id SAMEA5085962 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:19Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_62 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_62 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897315 SAMEA5085964 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:19Z External Id SAMEA5085964 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:19Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_64 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_64 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897316 SAMEA5085965 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:19Z External Id SAMEA5085965 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:19Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_65 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_65 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897317 SAMEA5085966 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:19Z External Id SAMEA5085966 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:19Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_66 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_66 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897318 SAMEA5085967 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:19Z External Id SAMEA5085967 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:19Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_67 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_67 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897319 SAMEA5085968 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:19Z External Id SAMEA5085968 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:19Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_68 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_68 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897321 SAMEA5085970 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:19Z External Id SAMEA5085970 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:19Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_7 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_7 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897322 SAMEA5085971 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:20Z External Id SAMEA5085971 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:20Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_70 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_70 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897323 SAMEA5085972 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:20Z External Id SAMEA5085972 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:20Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_71 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_71 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897324 SAMEA5085973 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:20Z External Id SAMEA5085973 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:20Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_72 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_72 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897325 SAMEA5085974 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:20Z External Id SAMEA5085974 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:20Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_73 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_73 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897326 SAMEA5085975 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:20Z External Id SAMEA5085975 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:20Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_74 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_74 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897327 SAMEA5085976 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:20Z External Id SAMEA5085976 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:20Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_75 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_75 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897328 SAMEA5085977 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:20Z External Id SAMEA5085977 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:20Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_76 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_76 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897329 SAMEA5085978 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:20Z External Id SAMEA5085978 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:20Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_77 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_77 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897331 SAMEA5085980 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:20Z External Id SAMEA5085980 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:20Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_79 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_79 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897332 SAMEA5085981 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:20Z External Id SAMEA5085981 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:20Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_8 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_8 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897333 SAMEA5085982 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:20Z External Id SAMEA5085982 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:20Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_80 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_80 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897334 SAMEA5085983 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:20Z External Id SAMEA5085983 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:20Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_81 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_81 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897335 SAMEA5085984 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:20Z External Id SAMEA5085984 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:20Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_82 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_82 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897337 SAMEA5085986 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:20Z External Id SAMEA5085986 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:20Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_84 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_84 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897338 SAMEA5085987 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:20Z External Id SAMEA5085987 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:20Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_85 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_85 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897339 SAMEA5085988 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:20Z External Id SAMEA5085988 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:20Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_86 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_86 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897340 SAMEA5085989 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:20Z External Id SAMEA5085989 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:20Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_87 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_87 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897341 SAMEA5085990 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:20Z External Id SAMEA5085990 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:20Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_88 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_88 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897342 SAMEA5085991 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:20Z External Id SAMEA5085991 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:20Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_89 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_89 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897343 SAMEA5085992 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:20Z External Id SAMEA5085992 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:20Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_9 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_9 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897344 SAMEA5085993 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:20Z External Id SAMEA5085993 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:20Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_90 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_90 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897345 SAMEA5085994 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:20Z External Id SAMEA5085994 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:20Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_91 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_91 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897346 SAMEA5085995 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:20Z External Id SAMEA5085995 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:20Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_92 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_92 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897348 SAMEA5085997 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:20Z External Id SAMEA5085997 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:20Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_94 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_94 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897349 SAMEA5085998 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:20Z External Id SAMEA5085998 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:20Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_95 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_95 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897350 SAMEA5085999 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:20Z External Id SAMEA5085999 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:20Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_96 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_96 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897351 SAMEA5086000 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:20Z External Id SAMEA5086000 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:20Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_1 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_1 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897352 SAMEA5086001 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:20Z External Id SAMEA5086001 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:20Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_10 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_10 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897354 SAMEA5086003 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:20Z External Id SAMEA5086003 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:20Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_12 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_12 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897355 SAMEA5086004 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:20Z External Id SAMEA5086004 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:20Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_13 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_13 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897356 SAMEA5086005 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:21Z External Id SAMEA5086005 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:21Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_14 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_14 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897357 SAMEA5086006 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:21Z External Id SAMEA5086006 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:21Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_15 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_15 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897358 SAMEA5086007 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:21Z External Id SAMEA5086007 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:21Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_16 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_16 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897359 SAMEA5086008 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:21Z External Id SAMEA5086008 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:21Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_17 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_17 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897360 SAMEA5086009 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:21Z External Id SAMEA5086009 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:21Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_18 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_18 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897361 SAMEA5086010 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:21Z External Id SAMEA5086010 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:21Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_19 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_19 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897362 SAMEA5086011 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:21Z External Id SAMEA5086011 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:21Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_2 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_2 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897364 SAMEA5086013 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:21Z External Id SAMEA5086013 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:21Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_21 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_21 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897365 SAMEA5086014 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:21Z External Id SAMEA5086014 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:21Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_22 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_22 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897366 SAMEA5086015 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:21Z External Id SAMEA5086015 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:21Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_23 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_23 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897367 SAMEA5086016 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:21Z External Id SAMEA5086016 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:21Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_24 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_24 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897368 SAMEA5086017 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:21Z External Id SAMEA5086017 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:21Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_25 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_25 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897370 SAMEA5086019 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:21Z External Id SAMEA5086019 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:21Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_27 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_27 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897371 SAMEA5086020 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:21Z External Id SAMEA5086020 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:21Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_28 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_28 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897372 SAMEA5086021 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:21Z External Id SAMEA5086021 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:21Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_29 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_29 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897373 SAMEA5086022 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:21Z External Id SAMEA5086022 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:21Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_3 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_3 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897374 SAMEA5086023 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:21Z External Id SAMEA5086023 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:21Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_30 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_30 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897375 SAMEA5086024 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:21Z External Id SAMEA5086024 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:21Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_31 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_31 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897376 SAMEA5086025 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:21Z External Id SAMEA5086025 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:21Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_32 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_32 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897377 SAMEA5086026 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:21Z External Id SAMEA5086026 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:21Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_33 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_33 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897378 SAMEA5086027 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:21Z External Id SAMEA5086027 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:21Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_34 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_34 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897379 SAMEA5086028 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:21Z External Id SAMEA5086028 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:21Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_35 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_35 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897381 SAMEA5086030 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:21Z External Id SAMEA5086030 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:21Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_37 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_37 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897382 SAMEA5086031 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:21Z External Id SAMEA5086031 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:21Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_38 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_38 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897383 SAMEA5086032 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:21Z External Id SAMEA5086032 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:21Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_39 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_39 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897384 SAMEA5086033 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:21Z External Id SAMEA5086033 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:21Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_4 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_4 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897385 SAMEA5086034 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:21Z External Id SAMEA5086034 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:21Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_40 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_40 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897387 SAMEA5086036 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:21Z External Id SAMEA5086036 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:21Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_42 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_42 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897388 SAMEA5086037 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:21Z External Id SAMEA5086037 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:21Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_43 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_43 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897389 SAMEA5086038 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:21Z External Id SAMEA5086038 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:21Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_44 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_44 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897390 SAMEA5086039 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:22Z External Id SAMEA5086039 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:22Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_45 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_45 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897391 SAMEA5086040 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:22Z External Id SAMEA5086040 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:22Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_46 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_46 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897392 SAMEA5086041 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:22Z External Id SAMEA5086041 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:22Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_47 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_47 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897393 SAMEA5086042 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:22Z External Id SAMEA5086042 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:22Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_48 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_48 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897394 SAMEA5086043 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:22Z External Id SAMEA5086043 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:22Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_49 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_49 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897395 SAMEA5086044 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:22Z External Id SAMEA5086044 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:22Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_5 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_5 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897397 SAMEA5086046 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:22Z External Id SAMEA5086046 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:22Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_51 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_51 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897398 SAMEA5086047 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:22Z External Id SAMEA5086047 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:22Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_52 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_52 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897399 SAMEA5086048 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:22Z External Id SAMEA5086048 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:22Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_53 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_53 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897400 SAMEA5086049 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:22Z External Id SAMEA5086049 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:22Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_54 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_54 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897401 SAMEA5086050 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:22Z External Id SAMEA5086050 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:22Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_55 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_55 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897403 SAMEA5086052 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:22Z External Id SAMEA5086052 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:22Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_57 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_57 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897404 SAMEA5086053 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:22Z External Id SAMEA5086053 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:22Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_58 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_58 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897405 SAMEA5086054 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:22Z External Id SAMEA5086054 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:22Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_59 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_59 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897406 SAMEA5086055 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:22Z External Id SAMEA5086055 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:22Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_6 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_6 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897407 SAMEA5086056 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:22Z External Id SAMEA5086056 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:22Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_60 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_60 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897408 SAMEA5086057 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:22Z External Id SAMEA5086057 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:22Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_61 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_61 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897409 SAMEA5086058 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:22Z External Id SAMEA5086058 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:22Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_62 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_62 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897410 SAMEA5086059 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:22Z External Id SAMEA5086059 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:22Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_63 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_63 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897411 SAMEA5086060 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:22Z External Id SAMEA5086060 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:22Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_64 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_64 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897412 SAMEA5086061 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:22Z External Id SAMEA5086061 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:22Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_65 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_65 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897215 SAMEA5085864 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:16Z External Id SAMEA5085864 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:16Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_60 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_60 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897221 SAMEA5085870 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:17Z External Id SAMEA5085870 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:17Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_66 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_66 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897231 SAMEA5085880 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:17Z External Id SAMEA5085880 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:17Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_75 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_75 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897237 SAMEA5085886 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:17Z External Id SAMEA5085886 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:17Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_80 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_80 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897248 SAMEA5085897 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:17Z External Id SAMEA5085897 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:17Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_90 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_90 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897254 SAMEA5085903 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:18Z External Id SAMEA5085903 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:18Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_2_96 broker name ArrayExpress common name fission yeast density 4 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_2_96 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 4 x 106 cells/ml in EMM at 32C ERS2897264 SAMEA5085913 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:18Z External Id SAMEA5085913 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:18Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_18 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_18 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897270 SAMEA5085919 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:18Z External Id SAMEA5085919 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:18Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_23 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_23 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897281 SAMEA5085930 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:18Z External Id SAMEA5085930 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:18Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_33 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_33 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897287 SAMEA5085936 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:19Z External Id SAMEA5085936 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:19Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_39 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_39 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897297 SAMEA5085946 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:19Z External Id SAMEA5085946 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:19Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_48 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_48 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897303 SAMEA5085952 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:19Z External Id SAMEA5085952 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:19Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_53 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_53 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897314 SAMEA5085963 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:19Z External Id SAMEA5085963 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:19Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_63 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_63 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897320 SAMEA5085969 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:19Z External Id SAMEA5085969 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:19Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_69 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_69 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897330 SAMEA5085979 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:20Z External Id SAMEA5085979 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:20Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_78 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_78 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897336 SAMEA5085985 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:20Z External Id SAMEA5085985 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:20Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_83 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_83 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897347 SAMEA5085996 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:20Z External Id SAMEA5085996 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:20Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_3_93 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_3_93 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897353 SAMEA5086002 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:20Z External Id SAMEA5086002 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:20Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_11 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_11 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897363 SAMEA5086012 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:21Z External Id SAMEA5086012 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:21Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_20 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_20 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897369 SAMEA5086018 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:21Z External Id SAMEA5086018 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:21Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_26 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_26 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897380 SAMEA5086029 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:21Z External Id SAMEA5086029 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:21Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_36 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_36 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897386 SAMEA5086035 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:21Z External Id SAMEA5086035 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:21Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_41 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_41 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897396 SAMEA5086045 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:22Z External Id SAMEA5086045 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:22Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_50 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_50 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897402 SAMEA5086051 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:22Z External Id SAMEA5086051 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:22Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_56 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_56 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897413 SAMEA5086062 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:22Z External Id SAMEA5086062 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:22Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_66 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_66 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897414 SAMEA5086063 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:22Z External Id SAMEA5086063 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:22Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_67 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_67 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897415 SAMEA5086064 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:22Z External Id SAMEA5086064 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:22Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_68 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_68 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897416 SAMEA5086065 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:22Z External Id SAMEA5086065 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:22Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_69 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_69 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897417 SAMEA5086066 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:22Z External Id SAMEA5086066 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:22Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_7 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_7 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897418 SAMEA5086067 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:22Z External Id SAMEA5086067 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:22Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_70 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_70 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897419 SAMEA5086068 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:22Z External Id SAMEA5086068 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:22Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_71 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_71 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897420 SAMEA5086069 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:22Z External Id SAMEA5086069 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:22Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_72 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_72 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897421 SAMEA5086070 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:22Z External Id SAMEA5086070 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:22Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_73 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_73 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897422 SAMEA5086071 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:22Z External Id SAMEA5086071 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:22Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_74 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_74 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897423 SAMEA5086072 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:22Z External Id SAMEA5086072 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:22Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_75 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_75 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897424 SAMEA5086073 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:01Z ENA-LAST-UPDATE 2018-11-13T18:38:22Z External Id SAMEA5086073 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:01Z INSDC last update 2018-11-13T18:38:22Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_76 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_76 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897425 SAMEA5086074 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:58Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086074 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:58Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_77 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_77 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897426 SAMEA5086075 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:58Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086075 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:58Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_78 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_78 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897427 SAMEA5086076 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:58Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086076 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:58Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_79 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_79 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897428 SAMEA5086077 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:58Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086077 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:58Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_8 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_8 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897429 SAMEA5086078 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:58Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086078 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:58Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_80 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_80 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897430 SAMEA5086079 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:58Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086079 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:58Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_81 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_81 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897431 SAMEA5086080 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:58Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086080 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:58Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_82 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_82 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897432 SAMEA5086081 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:58Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086081 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:58Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_83 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_83 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897433 SAMEA5086082 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:58Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086082 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:58Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_84 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_84 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897434 SAMEA5086083 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:58Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086083 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:58Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_85 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_85 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897435 SAMEA5086084 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:58Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086084 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:58Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_86 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_86 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897436 SAMEA5086085 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:58Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086085 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:58Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_87 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_87 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897437 SAMEA5086086 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:58Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086086 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:58Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_88 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_88 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897438 SAMEA5086087 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:58Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086087 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:58Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_89 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_89 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897439 SAMEA5086088 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:58Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086088 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:58Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_9 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_9 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897440 SAMEA5086089 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:58Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086089 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:58Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_90 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_90 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897441 SAMEA5086090 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:58Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086090 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:58Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_91 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_91 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897442 SAMEA5086091 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:58Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086091 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:58Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_92 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_92 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897443 SAMEA5086092 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:58Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086092 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:58Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_93 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_93 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897444 SAMEA5086093 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:58Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086093 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:58Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_94 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_94 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897445 SAMEA5086094 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:58Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086094 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:58Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_95 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_95 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897446 SAMEA5086095 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:58Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086095 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:58Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_4_96 broker name ArrayExpress common name fission yeast density 6 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_4_96 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 6 x 106 cells/ml in EMM at 32C ERS2897447 SAMEA5086096 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:58Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086096 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:58Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_1 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_1 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897448 SAMEA5086097 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:58Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086097 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:58Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_10 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_10 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897449 SAMEA5086098 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086098 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_11 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_11 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897450 SAMEA5086099 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086099 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_12 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_12 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897451 SAMEA5086100 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086100 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_13 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_13 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897452 SAMEA5086101 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086101 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_14 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_14 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897453 SAMEA5086102 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086102 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_15 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_15 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897454 SAMEA5086103 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086103 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_16 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_16 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897455 SAMEA5086104 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086104 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_17 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_17 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897456 SAMEA5086105 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086105 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_18 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_18 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897457 SAMEA5086106 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086106 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_19 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_19 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897458 SAMEA5086107 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086107 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_2 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_2 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897459 SAMEA5086108 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086108 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_20 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_20 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897460 SAMEA5086109 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:23Z External Id SAMEA5086109 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:23Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_21 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_21 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897461 SAMEA5086110 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:24Z External Id SAMEA5086110 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:24Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_22 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_22 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897463 SAMEA5086112 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:24Z External Id SAMEA5086112 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:24Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_24 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_24 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897464 SAMEA5086113 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:24Z External Id SAMEA5086113 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:24Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_25 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_25 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897465 SAMEA5086114 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:24Z External Id SAMEA5086114 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:24Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_26 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_26 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897466 SAMEA5086115 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:24Z External Id SAMEA5086115 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:24Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_27 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_27 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897467 SAMEA5086116 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:24Z External Id SAMEA5086116 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:24Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_28 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_28 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897468 SAMEA5086117 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:24Z External Id SAMEA5086117 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:24Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_29 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_29 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897469 SAMEA5086118 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:24Z External Id SAMEA5086118 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:24Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_3 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_3 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897470 SAMEA5086119 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:24Z External Id SAMEA5086119 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:24Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_30 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_30 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897471 SAMEA5086120 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:24Z External Id SAMEA5086120 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:24Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_31 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_31 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897472 SAMEA5086121 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:24Z External Id SAMEA5086121 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:24Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_32 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_32 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897473 SAMEA5086122 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:24Z External Id SAMEA5086122 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:24Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_33 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_33 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897474 SAMEA5086123 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:24Z External Id SAMEA5086123 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:24Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_34 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_34 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897475 SAMEA5086124 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:24Z External Id SAMEA5086124 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:24Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_35 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_35 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897476 SAMEA5086125 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:24Z External Id SAMEA5086125 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:24Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_36 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_36 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897477 SAMEA5086126 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:24Z External Id SAMEA5086126 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:24Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_37 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_37 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897478 SAMEA5086127 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:24Z External Id SAMEA5086127 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:24Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_38 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_38 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897479 SAMEA5086128 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:24Z External Id SAMEA5086128 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:24Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_39 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_39 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897480 SAMEA5086129 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:24Z External Id SAMEA5086129 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:24Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_4 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_4 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897481 SAMEA5086130 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:24Z External Id SAMEA5086130 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:24Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_40 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_40 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897482 SAMEA5086131 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:24Z External Id SAMEA5086131 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:24Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_41 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_41 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897483 SAMEA5086132 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:24Z External Id SAMEA5086132 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:24Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_42 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_42 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897484 SAMEA5086133 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:24Z External Id SAMEA5086133 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:24Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_43 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_43 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897485 SAMEA5086134 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:24Z External Id SAMEA5086134 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:24Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_44 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_44 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897486 SAMEA5086135 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:24Z External Id SAMEA5086135 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:24Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_45 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_45 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897487 SAMEA5086136 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:24Z External Id SAMEA5086136 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:24Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_46 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_46 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897488 SAMEA5086137 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:24Z External Id SAMEA5086137 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:24Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_47 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_47 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897489 SAMEA5086138 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:24Z External Id SAMEA5086138 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:24Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_48 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_48 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897490 SAMEA5086139 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:24Z External Id SAMEA5086139 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:24Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_49 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_49 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897491 SAMEA5086140 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:24Z External Id SAMEA5086140 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:24Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_5 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_5 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897492 SAMEA5086141 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:24Z External Id SAMEA5086141 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:24Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_50 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_50 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897493 SAMEA5086142 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:24Z External Id SAMEA5086142 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:24Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_51 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_51 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897494 SAMEA5086143 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:24Z External Id SAMEA5086143 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:24Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_52 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_52 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897495 SAMEA5086144 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:25Z External Id SAMEA5086144 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:25Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_53 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_53 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897496 SAMEA5086145 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:25Z External Id SAMEA5086145 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:25Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_54 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_54 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897497 SAMEA5086146 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:25Z External Id SAMEA5086146 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:25Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_55 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_55 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897498 SAMEA5086147 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:25Z External Id SAMEA5086147 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:25Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_56 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_56 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897499 SAMEA5086148 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:25Z External Id SAMEA5086148 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:25Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_57 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_57 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897500 SAMEA5086149 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:25Z External Id SAMEA5086149 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:25Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_58 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_58 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897501 SAMEA5086150 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:25Z External Id SAMEA5086150 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:25Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_59 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_59 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897502 SAMEA5086151 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:25Z External Id SAMEA5086151 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:25Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_6 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_6 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897503 SAMEA5086152 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:25Z External Id SAMEA5086152 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:25Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_60 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_60 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897504 SAMEA5086153 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:25Z External Id SAMEA5086153 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:25Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_61 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_61 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897505 SAMEA5086154 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:25Z External Id SAMEA5086154 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:25Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_62 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_62 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897506 SAMEA5086155 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:25Z External Id SAMEA5086155 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:25Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_63 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_63 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897507 SAMEA5086156 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:25Z External Id SAMEA5086156 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:25Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_64 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_64 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897508 SAMEA5086157 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:25Z External Id SAMEA5086157 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:25Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_65 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_65 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897509 SAMEA5086158 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:25Z External Id SAMEA5086158 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:25Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_66 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_66 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897510 SAMEA5086159 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:25Z External Id SAMEA5086159 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:25Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_67 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_67 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897511 SAMEA5086160 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:25Z External Id SAMEA5086160 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:25Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_68 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_68 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897512 SAMEA5086161 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:25Z External Id SAMEA5086161 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:25Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_69 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_69 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897513 SAMEA5086162 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:25Z External Id SAMEA5086162 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:25Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_7 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_7 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897514 SAMEA5086163 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:25Z External Id SAMEA5086163 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:25Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_70 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_70 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897515 SAMEA5086164 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:25Z External Id SAMEA5086164 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:25Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_71 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_71 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897516 SAMEA5086165 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:25Z External Id SAMEA5086165 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:25Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_72 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_72 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897517 SAMEA5086166 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:25Z External Id SAMEA5086166 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:25Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_73 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_73 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897518 SAMEA5086167 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:25Z External Id SAMEA5086167 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:25Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_74 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_74 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897519 SAMEA5086169 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:25Z External Id SAMEA5086169 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:25Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_75 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_75 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897520 SAMEA5086170 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:25Z External Id SAMEA5086170 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:25Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_76 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_76 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897521 SAMEA5086171 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:25Z External Id SAMEA5086171 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:25Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_77 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_77 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897522 SAMEA5086172 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:25Z External Id SAMEA5086172 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:25Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_78 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_78 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897523 SAMEA5086173 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:25Z External Id SAMEA5086173 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:25Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_79 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_79 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897524 SAMEA5086174 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:25Z External Id SAMEA5086174 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:25Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_8 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_8 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897525 SAMEA5086175 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:25Z External Id SAMEA5086175 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:25Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_80 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_80 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897526 SAMEA5086176 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:26Z External Id SAMEA5086176 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:26Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_81 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_81 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897527 SAMEA5086177 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:26Z External Id SAMEA5086177 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:26Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_82 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_82 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897529 SAMEA5086179 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:26Z External Id SAMEA5086179 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:26Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_84 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_84 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897530 SAMEA5086180 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:26Z External Id SAMEA5086180 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:26Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_85 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_85 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897531 SAMEA5086181 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:26Z External Id SAMEA5086181 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:26Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_86 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_86 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897532 SAMEA5086182 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:26Z External Id SAMEA5086182 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:26Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_87 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_87 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897533 SAMEA5086183 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:26Z External Id SAMEA5086183 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:26Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_88 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_88 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897535 SAMEA5086185 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:26Z External Id SAMEA5086185 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:26Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_9 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_9 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897536 SAMEA5086186 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:26Z External Id SAMEA5086186 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:26Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_90 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_90 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897537 SAMEA5086187 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:26Z External Id SAMEA5086187 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:26Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_91 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_91 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897538 SAMEA5086188 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:26Z External Id SAMEA5086188 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:26Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_92 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_92 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897539 SAMEA5086189 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:26Z External Id SAMEA5086189 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:26Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_93 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_93 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897540 SAMEA5086190 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:26Z External Id SAMEA5086190 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:26Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_94 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_94 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897541 SAMEA5086191 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:26Z External Id SAMEA5086191 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:26Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_95 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_95 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897542 SAMEA5086192 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:26Z External Id SAMEA5086192 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:26Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_96 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_96 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897543 SAMEA5086193 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:26Z External Id SAMEA5086193 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:26Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_1 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_1 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897544 SAMEA5086194 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:26Z External Id SAMEA5086194 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:26Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_10 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_10 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897546 SAMEA5086196 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:26Z External Id SAMEA5086196 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:26Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_12 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_12 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897547 SAMEA5086197 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:26Z External Id SAMEA5086197 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:26Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_13 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_13 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897548 SAMEA5086198 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:26Z External Id SAMEA5086198 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:26Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_14 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_14 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897549 SAMEA5086199 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:26Z External Id SAMEA5086199 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:26Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_15 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_15 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897550 SAMEA5086200 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:26Z External Id SAMEA5086200 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:26Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_16 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_16 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897552 SAMEA5086202 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:26Z External Id SAMEA5086202 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:26Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_18 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_18 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897553 SAMEA5086203 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:26Z External Id SAMEA5086203 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:26Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_19 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_19 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897554 SAMEA5086204 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:26Z External Id SAMEA5086204 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:26Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_2 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_2 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897555 SAMEA5086205 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:27Z External Id SAMEA5086205 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:27Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_20 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_20 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897556 SAMEA5086206 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:27Z External Id SAMEA5086206 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:27Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_21 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_21 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897557 SAMEA5086207 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:27Z External Id SAMEA5086207 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:27Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_22 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_22 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897558 SAMEA5086208 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:27Z External Id SAMEA5086208 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:27Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_23 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_23 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897559 SAMEA5086209 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:27Z External Id SAMEA5086209 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:27Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_24 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_24 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897560 SAMEA5086210 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:27Z External Id SAMEA5086210 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:27Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_25 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_25 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897562 SAMEA5086212 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:27Z External Id SAMEA5086212 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:27Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_27 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_27 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897563 SAMEA5086213 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:27Z External Id SAMEA5086213 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:27Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_28 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_28 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897564 SAMEA5086214 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:27Z External Id SAMEA5086214 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:27Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_29 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_29 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897565 SAMEA5086215 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:27Z External Id SAMEA5086215 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:27Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_3 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_3 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897566 SAMEA5086216 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:27Z External Id SAMEA5086216 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:27Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_30 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_30 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897568 SAMEA5086218 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:27Z External Id SAMEA5086218 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:27Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_32 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_32 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897569 SAMEA5086219 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:27Z External Id SAMEA5086219 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:27Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_33 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_33 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897570 SAMEA5086220 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:27Z External Id SAMEA5086220 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:27Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_34 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_34 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897571 SAMEA5086221 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:27Z External Id SAMEA5086221 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:27Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_35 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_35 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897572 SAMEA5086222 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:27Z External Id SAMEA5086222 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:27Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_36 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_36 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897573 SAMEA5086223 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:27Z External Id SAMEA5086223 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:27Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_37 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_37 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897574 SAMEA5086224 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:27Z External Id SAMEA5086224 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:27Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_38 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_38 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897575 SAMEA5086225 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:27Z External Id SAMEA5086225 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:27Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_39 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_39 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897576 SAMEA5086226 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:27Z External Id SAMEA5086226 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:27Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_4 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_4 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897577 SAMEA5086227 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:28Z External Id SAMEA5086227 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:28Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_40 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_40 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897579 SAMEA5086229 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:28Z External Id SAMEA5086229 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:28Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_42 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_42 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897580 SAMEA5086230 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:28Z External Id SAMEA5086230 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:28Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_43 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_43 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897581 SAMEA5086231 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:28Z External Id SAMEA5086231 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:28Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_44 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_44 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897582 SAMEA5086232 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:28Z External Id SAMEA5086232 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:28Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_45 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_45 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897583 SAMEA5086233 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:28Z External Id SAMEA5086233 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:28Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_46 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_46 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897585 SAMEA5086235 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:28Z External Id SAMEA5086235 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:28Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_48 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_48 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897586 SAMEA5086236 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:28Z External Id SAMEA5086236 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:28Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_49 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_49 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897587 SAMEA5086237 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:28Z External Id SAMEA5086237 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:28Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_5 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_5 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897588 SAMEA5086238 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:28Z External Id SAMEA5086238 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:28Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_50 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_50 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897589 SAMEA5086239 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:28Z External Id SAMEA5086239 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:28Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_51 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_51 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897590 SAMEA5086240 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:28Z External Id SAMEA5086240 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:28Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_52 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_52 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897591 SAMEA5086241 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:28Z External Id SAMEA5086241 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:28Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_53 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_53 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897592 SAMEA5086242 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:28Z External Id SAMEA5086242 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:28Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_54 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_54 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897593 SAMEA5086243 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:28Z External Id SAMEA5086243 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:28Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_55 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_55 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897595 SAMEA5086245 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:28Z External Id SAMEA5086245 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:28Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_57 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_57 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897596 SAMEA5086246 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:28Z External Id SAMEA5086246 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:28Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_58 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_58 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897597 SAMEA5086247 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:28Z External Id SAMEA5086247 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:28Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_59 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_59 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897598 SAMEA5086248 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:28Z External Id SAMEA5086248 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:28Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_6 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_6 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897599 SAMEA5086249 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:29Z External Id SAMEA5086249 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:29Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_60 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_60 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897601 SAMEA5086251 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:29Z External Id SAMEA5086251 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:29Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_62 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_62 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897602 SAMEA5086252 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:29Z External Id SAMEA5086252 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:29Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_63 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_63 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897603 SAMEA5086253 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:29Z External Id SAMEA5086253 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:29Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_64 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_64 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897604 SAMEA5086254 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:29Z External Id SAMEA5086254 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:29Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_65 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_65 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897605 SAMEA5086255 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:29Z External Id SAMEA5086255 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:29Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_66 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_66 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897606 SAMEA5086256 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:29Z External Id SAMEA5086256 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:29Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_67 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_67 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897607 SAMEA5086257 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:29Z External Id SAMEA5086257 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:29Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_68 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_68 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897608 SAMEA5086258 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:29Z External Id SAMEA5086258 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:29Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_69 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_69 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897609 SAMEA5086259 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:29Z External Id SAMEA5086259 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:29Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_7 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_7 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897610 SAMEA5086260 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:29Z External Id SAMEA5086260 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:29Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_70 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_70 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897612 SAMEA5086262 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:29Z External Id SAMEA5086262 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:29Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_72 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_72 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897613 SAMEA5086263 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:29Z External Id SAMEA5086263 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:29Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_73 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_73 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897614 SAMEA5086264 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:29Z External Id SAMEA5086264 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:29Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_74 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_74 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897615 SAMEA5086265 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:29Z External Id SAMEA5086265 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:29Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_75 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_75 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897616 SAMEA5086266 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:29Z External Id SAMEA5086266 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:29Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_76 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_76 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897618 SAMEA5086268 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:29Z External Id SAMEA5086268 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:29Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_78 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_78 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897619 SAMEA5086269 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:29Z External Id SAMEA5086269 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:29Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_79 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_79 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897620 SAMEA5086270 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:30Z External Id SAMEA5086270 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:30Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_8 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_8 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897621 SAMEA5086271 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:30Z External Id SAMEA5086271 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:30Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_80 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_80 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897622 SAMEA5086272 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:30Z External Id SAMEA5086272 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:30Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_81 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_81 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897623 SAMEA5086273 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:30Z External Id SAMEA5086273 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:30Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_82 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_82 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897624 SAMEA5086274 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:30Z External Id SAMEA5086274 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:30Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_83 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_83 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897625 SAMEA5086275 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:30Z External Id SAMEA5086275 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:30Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_84 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_84 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897626 SAMEA5086276 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:30Z External Id SAMEA5086276 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:30Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_85 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_85 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897628 SAMEA5086278 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:30Z External Id SAMEA5086278 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:30Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_87 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_87 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897629 SAMEA5086279 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:30Z External Id SAMEA5086279 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:30Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_88 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_88 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897630 SAMEA5086280 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:30Z External Id SAMEA5086280 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:30Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_89 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_89 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897631 SAMEA5086281 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:30Z External Id SAMEA5086281 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:30Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_9 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_9 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897632 SAMEA5086282 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:30Z External Id SAMEA5086282 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:30Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_90 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_90 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897634 SAMEA5086284 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:30Z External Id SAMEA5086284 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:30Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_92 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_92 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897635 SAMEA5086285 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:30Z External Id SAMEA5086285 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:30Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_93 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_93 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897636 SAMEA5086286 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:30Z External Id SAMEA5086286 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:30Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_94 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_94 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897637 SAMEA5086287 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:30Z External Id SAMEA5086287 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:30Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_95 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_95 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897638 SAMEA5086288 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:30Z External Id SAMEA5086288 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:30Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_96 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_96 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897639 SAMEA5086289 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:30Z External Id SAMEA5086289 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:30Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_7_1 broker name ArrayExpress common name fission yeast density 10 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_7_1 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 10 x 106 cells/ml in EMM at 32C ERS2897640 SAMEA5086290 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:30Z External Id SAMEA5086290 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:30Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_7_10 broker name ArrayExpress common name fission yeast density 10 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_7_10 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 10 x 106 cells/ml in EMM at 32C ERS2897641 SAMEA5086291 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:30Z External Id SAMEA5086291 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:30Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_7_11 broker name ArrayExpress common name fission yeast density 10 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_7_11 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 10 x 106 cells/ml in EMM at 32C ERS2897642 SAMEA5086292 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:30Z External Id SAMEA5086292 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:30Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_7_12 broker name ArrayExpress common name fission yeast density 10 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_7_12 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 10 x 106 cells/ml in EMM at 32C ERS2897643 SAMEA5086293 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:30Z External Id SAMEA5086293 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:30Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_7_13 broker name ArrayExpress common name fission yeast density 10 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_7_13 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 10 x 106 cells/ml in EMM at 32C ERS2897645 SAMEA5086295 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:31Z External Id SAMEA5086295 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:31Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_7_15 broker name ArrayExpress common name fission yeast density 10 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_7_15 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 10 x 106 cells/ml in EMM at 32C ERS2897646 SAMEA5086296 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:31Z External Id SAMEA5086296 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:31Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_7_16 broker name ArrayExpress common name fission yeast density 10 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_7_16 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 10 x 106 cells/ml in EMM at 32C ERS2897647 SAMEA5086297 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:31Z External Id SAMEA5086297 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:31Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_7_17 broker name ArrayExpress common name fission yeast density 10 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_7_17 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 10 x 106 cells/ml in EMM at 32C ERS2897648 SAMEA5086298 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:31Z External Id SAMEA5086298 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:31Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_7_18 broker name ArrayExpress common name fission yeast density 10 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_7_18 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 10 x 106 cells/ml in EMM at 32C ERS2897649 SAMEA5086299 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:31Z External Id SAMEA5086299 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:31Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_7_19 broker name ArrayExpress common name fission yeast density 10 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_7_19 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 10 x 106 cells/ml in EMM at 32C ERS2897650 SAMEA5086300 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:31Z External Id SAMEA5086300 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:31Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_7_2 broker name ArrayExpress common name fission yeast density 10 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_7_2 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 10 x 106 cells/ml in EMM at 32C ERS2897651 SAMEA5086301 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:31Z External Id SAMEA5086301 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:31Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_7_20 broker name ArrayExpress common name fission yeast density 10 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_7_20 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 10 x 106 cells/ml in EMM at 32C ERS2897652 SAMEA5086302 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:31Z External Id SAMEA5086302 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:31Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_7_21 broker name ArrayExpress common name fission yeast density 10 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_7_21 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 10 x 106 cells/ml in EMM at 32C ERS2897653 SAMEA5086303 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:31Z External Id SAMEA5086303 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:31Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_7_22 broker name ArrayExpress common name fission yeast density 10 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_7_22 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 10 x 106 cells/ml in EMM at 32C ERS2897654 SAMEA5086304 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:31Z External Id SAMEA5086304 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:31Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_7_23 broker name ArrayExpress common name fission yeast density 10 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_7_23 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 10 x 106 cells/ml in EMM at 32C ERS2897656 SAMEA5086306 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:31Z External Id SAMEA5086306 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:31Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_7_25 broker name ArrayExpress common name fission yeast density 10 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_7_25 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 10 x 106 cells/ml in EMM at 32C ERS2897657 SAMEA5086307 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:31Z External Id SAMEA5086307 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:31Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_7_26 broker name ArrayExpress common name fission yeast density 10 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_7_26 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 10 x 106 cells/ml in EMM at 32C ERS2897658 SAMEA5086308 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:31Z External Id SAMEA5086308 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:31Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_7_27 broker name ArrayExpress common name fission yeast density 10 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_7_27 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 10 x 106 cells/ml in EMM at 32C ERS2897659 SAMEA5086309 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:31Z External Id SAMEA5086309 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:31Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_7_28 broker name ArrayExpress common name fission yeast density 10 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_7_28 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 10 x 106 cells/ml in EMM at 32C ERS2897660 SAMEA5086310 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:31Z External Id SAMEA5086310 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:31Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_7_29 broker name ArrayExpress common name fission yeast density 10 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_7_29 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 10 x 106 cells/ml in EMM at 32C ERS2897662 SAMEA5086312 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:31Z External Id SAMEA5086312 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:31Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_7_30 broker name ArrayExpress common name fission yeast density 10 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_7_30 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 10 x 106 cells/ml in EMM at 32C ERS2897663 SAMEA5086313 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:31Z External Id SAMEA5086313 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:31Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_7_31 broker name ArrayExpress common name fission yeast density 10 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_7_31 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 10 x 106 cells/ml in EMM at 32C ERS2897664 SAMEA5086314 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:31Z External Id SAMEA5086314 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:31Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_7_32 broker name ArrayExpress common name fission yeast density 10 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_7_32 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 10 x 106 cells/ml in EMM at 32C ERS2897665 SAMEA5086315 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:31Z External Id SAMEA5086315 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:31Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_7_33 broker name ArrayExpress common name fission yeast density 10 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_7_33 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 10 x 106 cells/ml in EMM at 32C ERS2897666 SAMEA5086316 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:31Z External Id SAMEA5086316 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:31Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_7_34 broker name ArrayExpress common name fission yeast density 10 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_7_34 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 10 x 106 cells/ml in EMM at 32C ERS2897667 SAMEA5086317 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:31Z External Id SAMEA5086317 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:31Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_7_35 broker name ArrayExpress common name fission yeast density 10 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_7_35 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 10 x 106 cells/ml in EMM at 32C ERS2897668 SAMEA5086318 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:31Z External Id SAMEA5086318 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:31Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_7_36 broker name ArrayExpress common name fission yeast density 10 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_7_36 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 10 x 106 cells/ml in EMM at 32C ERS2897669 SAMEA5086319 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:31Z External Id SAMEA5086319 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:31Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_7_37 broker name ArrayExpress common name fission yeast density 10 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_7_37 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 10 x 106 cells/ml in EMM at 32C ERS2897462 SAMEA5086111 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:24Z External Id SAMEA5086111 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:24Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_23 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_23 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897528 SAMEA5086178 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:26Z External Id SAMEA5086178 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:26Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_83 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_83 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897534 SAMEA5086184 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:26Z External Id SAMEA5086184 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:26Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_5_89 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_5_89 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897545 SAMEA5086195 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:26Z External Id SAMEA5086195 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:26Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_11 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_11 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897551 SAMEA5086201 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:26Z External Id SAMEA5086201 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:26Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_17 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_17 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897561 SAMEA5086211 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:27Z External Id SAMEA5086211 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:27Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_26 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_26 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897567 SAMEA5086217 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:02:59Z ENA-LAST-UPDATE 2018-11-13T18:38:27Z External Id SAMEA5086217 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:02:59Z INSDC last update 2018-11-13T18:38:27Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_31 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_31 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897578 SAMEA5086228 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:28Z External Id SAMEA5086228 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:28Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_41 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_41 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897584 SAMEA5086234 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:28Z External Id SAMEA5086234 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:28Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_47 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_47 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897594 SAMEA5086244 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:28Z External Id SAMEA5086244 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:28Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_56 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_56 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897600 SAMEA5086250 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:29Z External Id SAMEA5086250 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:29Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_61 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_61 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897611 SAMEA5086261 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:29Z External Id SAMEA5086261 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:29Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_71 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_71 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897617 SAMEA5086267 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:29Z External Id SAMEA5086267 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:29Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_77 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_77 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897627 SAMEA5086277 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:30Z External Id SAMEA5086277 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:30Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_86 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_86 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897633 SAMEA5086283 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:30Z External Id SAMEA5086283 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:30Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_6_91 broker name ArrayExpress common name fission yeast density 8 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_6_91 scientific_name Schizosaccharomyces pombe spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 8 x 106 cells/ml in EMM at 32C ERS2897644 SAMEA5086294 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:31Z External Id SAMEA5086294 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:31Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_7_14 broker name ArrayExpress common name fission yeast density 10 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_7_14 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 10 x 106 cells/ml in EMM at 32C ERS2897655 SAMEA5086305 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:31Z External Id SAMEA5086305 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:31Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_7_24 broker name ArrayExpress common name fission yeast density 10 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_7_24 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 10 x 106 cells/ml in EMM at 32C ERS2897661 SAMEA5086311 4896 Schizosaccharomyces pombe Protocols: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells. 972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting. Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3-5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced. ENA-FIRST-PUBLIC 2018-11-14T17:03:00Z ENA-LAST-UPDATE 2018-11-13T18:38:31Z External Id SAMEA5086311 INSDC center name MRC London Institute of Medical Sciences and Institute of Clinical Sciences (ICS, Imperial College London) INSDC first public 2018-11-14T17:03:00Z INSDC last update 2018-11-13T18:38:31Z INSDC status public Submitter Id E-MTAB-68251542132175:2502_7_3 broker name ArrayExpress common name fission yeast density 10 x 106 genotype wild type genotype growth condition Flask medium EMM2 sample name E-MTAB-68251542132175:2502_7_3 scientific_name Schizosaccharomyces pombe single cell well quality 1 cell spike in ERCC spike in set A spike in dilution 1l of 1:106 dilution strain 972h- temperature 32 treatment Proliferation, 10 x 106 cells/ml in EMM at 32C