GSM3417891: OX-yedQ-3; Pseudomonas syringae pv. syringae B728a; RNA-Seq
To test the effect of high c-di-GMP level in Psy B728a on the transcriptome, cells were recovered and cultured to early stationary (OD600 ~ 2) in NYGB medium consisting of 8 g nutrient broth, 10 g glucose and 5 g yeast extract each liter. Cells were mixed with bacterial RNA Protect Reagent (Qiagen) to keep intact RNA. Total RNA was purified using RNeasy Mini Kit (Qiagen). RNase-free DNase Set (Qiagen) was applied to remove DNA contaminant through on-column DNase digestion. Second DNA removal step was applied using Ambion™ TURBO DNA-free Kit. rRNA was depleted using Ribo-Zero rRNA removal Kit (Illumina). The integrity and concentration of total RNA and DNA contamination were examined by Agilent TapeStation System (Agilent Technologies) and Qubit® 2.0 Fluorometer (Invitrogen). Reverse transcription to cDNA was done using NEBNext RNA first and second strand synthesis module (NEB). Analysis of gene expression was carried out via Illumina RNA-Seq. Libraries were produced using an Illumina TruSeq Stranded messenger RNA Sample Prep Kit. The libraries were sequenced using Illumina HiSeq 2500 platform with a paired-end protocol and read lengths of 100 nt. TruSeq RNA sample prep kit protocol (Illumina)