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Nanopore RNA-seq of P. lutheri culture with oil
DNA extraction was done using PowerSoil DNA isolation kit (MO BIO) according to manufacturers instructions.  The DNA samples from each replicate experimental set were pooled to prepare the one DNA sample per each variant. An aliquot of each DNA samples was checked for quality and quantity by gel electrophoresis on the 0.8% agarose gel and by measuring the concentration of DNA by Qubit dsDNA HS kit (Life Technology) with Qubit Fluorometer. These DNA samples were used as a template for barcoded-amplicon sequencing. To amplify and sequence the full-length 16S rRNA gene we used 16S Barcoding kit (SQK-RAB204) according to the protocol by Oxford Nanopore Technologies. The obtained PCR products were purified using Agencourt AMPure XP beads (Beckman Coulter) according to the Nanopores protocol. The purified and pooled in equimolar proportion DNA was subjected to library construction by attachment of rapid 1D sequencing adapters provided in 16S Barcoding kit (Oxford Nanopore). After the priming the Flow Cell (FLO-MIN106 R9 Version), the prepared library was loaded into MinIONTM Flow Cells and sequenced during 24 hours on MinIONTM using MinKNOWTM workflow.
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SUB6615396
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