RNA-Seq Library Preparation and Sequencing

Total RNA was isolated, ribosomal RNA was removed, and mRNA libraries were generated with the ScriptSeq V2 RNA-Seq Complete Gold Kit for Epidemiology (Epicenter). cDNA samples were pooled at equal nanomolar concentration and 150-base paired-end reads were generated on the Illumina NextSeq instrument. Sequences from each sample were trimmed of adapters using Trimmomatic [48 ]. Primer, rRNA, tRNA, phiX, and human contaminant sequences were removed using the Burrows-Wheeler Aligner (BWA) [49 (link)]. The resulting reads were termed clean and were used for further taxonomic and metabolic function analysis.