title: Illumina HiSeq 2500 paired end sequencing; Single-cell phenotyping and RNA sequencing reveal novel patterns of gene expression heterogeneity and regulation during growth and stress adaptation in a unicellular eukaryote
design: Single-cell phenotyping and RNA sequencing reveal novel patterns of gene expression heterogeneity and regulation during growth and stress adaptation in a unicellular eukaryote
library_construction_protocol: scRB-seq experiment. Single cells were imaged with a 20x objective on a Singer MSM-400 tetrad-dissection microscope (Singer Instruments), picked into 3µl of QuickExtract™ for RNA extraction solution (Lucigen, Epicenter) in 200µl PCR tubes and immediately snap frozen at -80°C. For RNA samples 3pg of total RNA isolated from matching culture by hot phenol extraction were processed as single cells.  972h- fission yeast cells were cultured with a seeding density of 0.5 x 106 cells/ml in all experiments in EMM2 medium unless stated otherwise. Heat stress: cells were grown in YE medium at 25°C up to a density of 2-4 x 106 cells/ml. The cells were transferred to a water bath maintained at 37°C or 39°C (for datasets, 1712_1, 1712_2, 0408_2 (“Heat”)). For studying recovery from heat shock, cells were transferred post heat-shock to a turbidostat maintained at density of 4 x 106 cells/ml and a temperature of either 25°C or 37°C. Glycerol growth: cells were grown in YE medium with 3% glycerol and 0.1% glucose. Osmotic shock, Oxidative stress: Cells were cultured in YE medium up to a density of 4 x 106 cells/ml. To induce osmotic shock, an equal volume of 2M sorbitol prepared in YE medium to a final concentration of 1M sorbitol. To induce oxidative stress, cells were treated with 0.5mM H2O2 for 15 or 60 min. Nitrogen starvation: Cells were pre-cultured in EMM2 medium with NH4Cl as nitrogen source up to a density of 2 x 106 cells/ml. Cells were harvested by centrifugation, washed twice with EMM medium without nitrogen and re-suspended in medium without nitrogen. Cells were harvested at 6 and 24 hours after starvation. All cultures were snap frozen at the time of harvesting.  Single cells were lysed at 98°C for 10 min in a PCR machine. Total RNA was extracted using hot phenol. Single cells were lysed at 98°C for 10 min in a PCR machine, and library preparation performed based on3–5, using primer sequences described in3. The protocol was modified as follows. Briefly, oligo dT containing cell indexes 1-96 and UMIs were added to each well at a final concentration of 1µM. Primers were annealed to the RNA template at 72°C for 3min, and components for reverse transcription added with final concentrations of 100U Superscript II reverse transcriptase (Invitrogen), 10U RNAse inhibitor (Invitrogen), 1x Superscript buffer, 5mM DTT, 1M Betaine (Sigma), 1.5mM MgCl2, 1mM of each dNTPs, 1/106 dilution of ERCC spikes Set A (NEB), 1µM RNA-TSO primer (As in Soumillon et al but fully RNA). Reverse transcription was carried out at 42°C for 90min, after which the temperature was ramped between 50°C and 42°C for 10 cycles of 2min each. The reaction was heat inactivated at 70°C for 15min and the reaction cooled to 15°C. Each single cell sample was treated with 20U Exonuclease I (NEB) for 30min at 37°C followed by heat inactivation at 80°C for 20min. Sets of 96 samples were pooled and purified using a PCR purification kit (Qiagen) and eluted in 60µl elution buffer (EB) containing 10mM Tris-Cl, pH 7.5. Samples were treated with 40U of Exonuclease I for 30min at 37°C for a second time followed by heat inactivation at 80°C for 20min. PCR was performed on the pooled sample adding 1x KAPA HiFi buffer, 0.075mM of each dNTPs, 1µM PCR primer, and 1.25U KAPA HiFi enzyme. PCR cycling was done with denaturation at 98°C for 3min, followed by 25 cycles of denaturation, annealing and extension at 98°C, 60°C and 72°C for 20s, 15s and 1min respectively. A final extension of 72°C for 5min was done before cooling the samples at 15°C. Samples were purified using 0.6x Agencourt beads and eluted in 10-15µl of nuclease free 10mM Tris pH 7.5. Libraries were quantified on an Agilent Bioanalyser using an HS-DNA chip to confirm the presence of a clean peak at ~1000bp. Between 1-2ng of PCR library was used for tagmentation using the Illumina Nextera XT kit using a modified I5 primer as described in3,4. Between 8 and 12 PCR cycles were performed post tagmentation to amplify the 3' fragments carrying the poly-A tail, the cell barcode and the UMI. The final libraries were purified using Agencourt beads twice at 1x bead concentration and final elution done in elution buffer. Libraries were quantified using on a Bioanalyser and sequenced.
attributes.tag: Experimental Factor: growth condition
attributes.value: Flask
attributes.tag: Experimental Factor: single cell identifier
attributes.value: 2911_1_95