strain/background: DFL12 light/dark condition: light days in co-culture with p. minimum: 12
Treatment protocol
For transcriptome analysis of the co-culture in the light and in the dark, sampling was achieved at day 12, as the algae reached the late exponential growth phase. To the time point 5 hours growth in the light or in the dark, 50 ml cultures were pelleted in 2 ml EPs and covered with 1 ml Trizol reagent and then snap frozen in liquid nitrogen and stored at -70° C until RNA isolation.
Growth protocol
Co-cultures were obtained by adding 10E7 cells ml-1 bacteria cells to cultures of P. minimum immediately after subculturing them in fresh medium with an initial density of approximately 2000 cells ml-1. Cultures were grown in 100-ml batches in 300-ml Erlenmeyer flasks at 22°C under a 12:12 h light-dark cycle with a light intensity of about 40 µmol photons m-2s-1.
Extracted molecule
total RNA
Extraction protocol
RNA isolation: For RNA extraction, the samples were thawed at room temperature then transferred to a cryotube filled with 0.3 g acid-washed glass beads. The cells were homogenized using FastPrep-24 instrument (MP Biomedicals, California, USA) at 6.0 m/s for 3 min and then incubated for 5 min at room temperature. Samples were centrifuged at 12,000 x g for 10 min at 4°C and the supernatants were transferred to fresh tubes, followed by the addition of 100 µl of 1-bromo-3-chloropropane (BCP, Sigma, Germany) and incubation for 10 min at room temperature. Samples were centrifuged at 12,000 x g for 10 min at 4°C, after which the aqueous phase was transferred to new tubes and mixed with 500 µl of absolute ethanol. Extracts were applied to RNeasy spin columns (RNeasy mini kit, Qiagen, Hilden, Germany) and processed according to the manufacturer's instructions. In addition, samples were treated with DNase I (Qiagen, Hilden, Germany). Removal of genomic DNA was verified via PCR. The concentration of the RNA was quantified using a NanoDrop spectrophotometer (Peqlab, Erlangen, Germany) and the RNA integrity was assessed using Bioanalyzer 2100 (Agilent, Santa Clara, USA). Ribosomal RNA depletion: Oligotex mRNA kit (Qiagen, Hilden, Germany) was used to isolate the algal mRNA. For isolation of D. shibae mRNA, MICROBEnrich kit (Ambion, Life Technologies, Darmstadt, Germany) was used to remove the eukaryotic rRNA and MICROBExpress kit (Ambion, Life Technologies, Darmstadt, Germany) to remove the prokaryotic rRNA, according to the manufacturer's instructions.
Label
Cy3
Label protocol
2 µg RNA were labelled with Cy3 or Cy5 using the Universal Linker System (Kreatech) according to the manufacturer's protocol.
strain/background: DFL12 light/dark condition: dark days in co-culture with p. minimum: 12
Treatment protocol
For transcriptome analysis of the co-culture in the light and in the dark, sampling was achieved at day 12, as the algae reached the late exponential growth phase. To the time point 5 hours growth in the light or in the dark, 50 ml cultures were pelleted in 2 ml EPs and covered with 1 ml Trizol reagent and then snap frozen in liquid nitrogen and stored at -70° C until RNA isolation.
Growth protocol
Co-cultures were obtained by adding 10E7 cells ml-1 bacteria cells to cultures of P. minimum immediately after subculturing them in fresh medium with an initial density of approximately 2000 cells ml-1. Cultures were grown in 100-ml batches in 300-ml Erlenmeyer flasks at 22°C under a 12:12 h light-dark cycle with a light intensity of about 40 µmol photons m-2s-1.
Extracted molecule
total RNA
Extraction protocol
RNA isolation: For RNA extraction, the samples were thawed at room temperature then transferred to a cryotube filled with 0.3 g acid-washed glass beads. The cells were homogenized using FastPrep-24 instrument (MP Biomedicals, California, USA) at 6.0 m/s for 3 min and then incubated for 5 min at room temperature. Samples were centrifuged at 12,000 x g for 10 min at 4°C and the supernatants were transferred to fresh tubes, followed by the addition of 100 µl of 1-bromo-3-chloropropane (BCP, Sigma, Germany) and incubation for 10 min at room temperature. Samples were centrifuged at 12,000 x g for 10 min at 4°C, after which the aqueous phase was transferred to new tubes and mixed with 500 µl of absolute ethanol. Extracts were applied to RNeasy spin columns (RNeasy mini kit, Qiagen, Hilden, Germany) and processed according to the manufacturer's instructions. In addition, samples were treated with DNase I (Qiagen, Hilden, Germany). Removal of genomic DNA was verified via PCR. The concentration of the RNA was quantified using a NanoDrop spectrophotometer (Peqlab, Erlangen, Germany) and the RNA integrity was assessed using Bioanalyzer 2100 (Agilent, Santa Clara, USA). Ribosomal RNA depletion: Oligotex mRNA kit (Qiagen, Hilden, Germany) was used to isolate the algal mRNA. For isolation of D. shibae mRNA, MICROBEnrich kit (Ambion, Life Technologies, Darmstadt, Germany) was used to remove the eukaryotic rRNA and MICROBExpress kit (Ambion, Life Technologies, Darmstadt, Germany) to remove the prokaryotic rRNA, according to the manufacturer's instructions.
Label
Cy5
Label protocol
2 µg RNA were labelled with Cy3 or Cy5 using the Universal Linker System (Kreatech) according to the manufacturer's protocol.
Hybridization protocol
Hybridization of 500 ng labelled RNA was performed according to the Agilent two-color microarray protocol.
Scan protocol
Microarrays were scanned using the Agilent DNA microarray scanner. Raw data was collected using the Agilent Feature Extraction software v10.7.
Data processing
Median values of Cy3 and Cy5 channels were processed in the R environment using the limma package. Background correction was performed using the normexp method, Cy3 and Cy5 channels were loess normalized, Quantile-normalization was used for separate microarrays.
