title: GSM1302413: D. shibae 5 h in the light after 12 days in co-culture with the dinoflagellate P. minimum, replicate 2; Dinoroseobacter shibae DFL 12 = DSM 16493; RNA-Seq
library_construction_protocol: RNA isolation: For RNA extraction, the sample was thawed at room temperature and then transferred to a cryotube filled with 0.3 g acid-washed glass beads. The cells were homogenized using the 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 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 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 DNAae I (Qiagen, Hilden, Germany). Removal of genomic DNA was verified via PCR using total RNA as template. 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: The Oligotex mRNA kit (Qiagen, Hilden, Germany) was used to isolate the algal mRNA. For isolation of D. shibae mRNA, the 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. Libraries of 300 bp for RNA sequencing were prepared using the mRNA-Seq sample preparation kit (Illumina, San Diego, CA, USA) according to the manufacturer's instructions. Quality control of the prepared libraries was validated using the Agilent Bioanalyzer (Agilent Technologies, Santa Clara, USA). Cluster generation was performed using the Illumina cluster station.
attributes.tag: GEO Accession
attributes.value: GSM1302413