title: RNA-Seq of Pseadomonas aeruginosa in a polymicrobial setting in the presence of arachidonic acid
design: Sequencing libraries will be prepared using the NEXTflex Rapid RNA-Seq kit using rRNA-depleted RNA. Each RNA sample will be assigned a specific NEXTflex RNA barcode. The libraries will be quantified using the Qubit DNA HS Assay and fragment size distribution of each library will be determined on the Bioanalyzer (Agilent). Libraries will be normalized to a concentration of 4nM with 10 mM Tris-HCl (pH 8.5) and subsequently combined at equal volumes to obtain an equimolar library pool. The final, pooled library will be quantified using the Qubit DNA HS Assay and the con-centration of adaptor-ligated DNA molecules will be confirmed through qPCR-based absolute quantification using the Roche Library Quantification Kit. The average frag-ment size will be determined on the Bioanalyzer (Agilent). These values are required to calculate the final nanomolar (nM) concentration prior to sequencing. Following denaturation, the pooled library will be combined with the PhiX positive control at a spike-in concentration of 1% v/v. The pooled library + 1% PhiX will be diluted to a final concentration of 1.5pM before setting up the sequencing reaction. Sequencing will be performed on the Illumina NextSeq 500 or NextSeq 550 instrument using NextSeq 500/550 Mid Output Reagent Kit v2.5 chemistry (150 cycle).