title: RNA-Seq of Pseudomonas aeruginosa: dual species biofilm with Streptococcus salivarius
design: RNA was purified using the Direct-zol RNA Mini Prep kit (Zymo Research) according to the manufacturers instructions. mRNA-sequencing was performed at the UAB Heflin Center for Genomic Sciences, using an Illumina NextSeq 500 as described by the manufacturer (Illumina, Inc). The Agilent SureSelect Strand Specific mRNA library kit (Agilent) and ribosome reduction with the RiboMinus protocol for Gram-negative and Gram-positive bacteria (Life Technologies) were used as described by the manufacturer. The resulting mRNA was randomly fragmented with cations and heat, followed by first strand synthesis using random primers. Second strand cDNA production was done with standard techniques, and cDNA libraries were quantitated using qPCR in a Roche LightCycler 480 with the Kapa Biosystems kit for Illumina library quantitation (Kapa Biosystems) prior to cluster generation, which was performed according to the manufacturers recommendations for onboard clustering (Illumina).