Transcriptome Analysis of Stenotrophomonas maltophilia

Overnight-cultured bacteria cultures were inoculated into fresh LB broth with an initial OD_450nm of 0.15. All cultures were grown at 37°C for 5 h and DNA-free RNA was extracted as described previously [11 (link)]. Ribosomal RNA (rRNA) was depleted from 5 μg of total RNA using the Ribo-Zero rRNA Removal Kit for bacteria (Epicentre, USA). The sequencing library for mRNA-seq was prepared according to the protocol for the "TruSeq RNA sample preparation” (Illumina Inc., USA). Briefly, the rRNA depleted mRNA was fragmented, and first-strand cDNA was synthesized using random hexamers following by second-strand cDNA synthesis, end repair, addition of a single A base and adapter ligation. The adapter-ligated cDNA library was amplified by PCR for 6 cycles using KAPA HiFi DNA polymerase (Kapa Biosystems). The enriched cDNA library was sequenced on a MiSeq (Illumina Inc., USA) using 250 bp paired-end reads. After trimming of low quality of bases (< Q30), the first 12 bases and adapters, the trimmed Reads were mapped to the Stenotrophomonas maltophilia K279a genome (GenBank acc. no. NC_010943.1) and run RNA-seq analysis by CLC Genomics Workbench v 6.0 (CLC Bio).