title: GSM8805582: WT, VA + Glucose, Replicate 2; Novosphingobium aromaticivorans DSM 12444; RNA-Seq
library_construction_protocol: The RNA was extracted with acid phenol:chloroform, following the manufacture´s instructions (Thermo Fisher Scientific; Waltham, USA). The cells were pelleted by centrifugation (4,300 x g for 10 minutes) at 4°C and stored at -80°C. Cell pellets were resuspended in 1 mL of lysis solution (2 % SDS, 16 mM EDTA) and heated at 65˚C for 5 minutes. Samples were mixed with 1 mL of hot acid phenol:chloroform and incubated at 65˚C for 5 minutes. To separate the RNA from other biomolecules, the samples were centrifuged at room temperature for 15 minutes at 20,000 g. The aqueous phase was transferred to a new tube and washed two more times with hot acid phenol:chloroform. The aqueous phase was mixed with 1 mL of chloroform and centrifuged for 15 minutes at 20,000 g. To precipitate the RNA, the samples were mixed with 100 µL of 3 M sodium acetate and 1 mL of isopropanol and incubated at -20oC for at least 1 h. Samples were centrifugated at 4˚C for 30 minutes at 16,000 g. The pellet was washed twice with 1 mL of cold 75 % EtOH. Once residual ethanol was removed by pipetting and evaporation, the pellet was resuspended in 40 μL of RNase-free water. Remaining DNA was removed with the TURBO DNA-free Kit (Thermo Fisher Scientific; Waltham, MA, USA), according to the manufacturer´s instructions. Finally, the RNA was cleaned using the RNeasy Mini Kit (Qiagen, Hilden, Germany), following the manufacturer's recommendations. RNA-seq library preparation and sequencing was performed by the Joint Genome Institute (JGI) using default parameters. rRNA in the samples was depleted using the QIAseq FastSelect kit (Qiagen, Hilden, Germany). Libraries were constructed using the TruSeq stranded mRNA kit (Illumina) following standard JGI protocols.
identifiers.namespace: GEO
identifiers.id: GSM8805582_r1