Ecological perturbations impact species-specific gene expression of the oral microbiota
Preparation of DNA and RNA library was performed according to Illuminas TruSeq Nano DNA Sample Preparation protocol and Stranded mRNA, respectively. DNA samples were sheared on a Covaris E220 to ~450bp, following the manufacturers recommendation, and uniquely tagged with one of Illuminas TruSeq HT DNA dual barcode combination to enable sample pooling for sequencing. The following modifications were applied to Illuminas TruSeq Stranded mRNA protocol. The oligo-dT mRNA purification step was omitted and instead, 200ng of total RNA were directly added to the Elution2-Frag-Prime step. The PCR amplification step, which selectively enriches for library fragments that have adapters ligated on both ends, was performed according to the manufacturers recommendation but the number of amplification cycles was reduced to 12. Each library was uniquely tagged with one of Illuminas TruSeq HT RNA dual barcode combination to allow pooling of libraries for sequencing. Both DNA and RNA libraries were quantitated using Promega's QuantiFluor dsDNA assay and the average library size was determined on an Agilent Tapestation 4200. Library concentrations were then normalized to 4nM and validated by qPCR on a QuantStudio-3 real-time PCR system (Applied Biosystems), using the Kapa library quantification kit for Illumina platforms (Kapa Biosystems).