title: GSM2977618: Flg22 5hpi Rep3; Pseudomonas syringae pv. tomato str. DC3000; RNA-Seq
library_construction_protocol: Leaves from each treatment were arranged on two sheets of parafilm, rolled and inserted into the barrel of a two 20 mL syringes. A RNAlater-like RNA stabilizing buffer pH 5.2, was poured into each syringe, sealed, and vacuum infiltrated at 95 kPa for 2 min followed by a slow release of the vacuum.  Vacuum-infiltration with RNA stabilizing buffer was conducted twice on inoculated leaves. Excess RNA stabilizing buffer was decanted and the syringes were placed into 50 mL conical tubes and centrifuged at 1000 g for 10 min at 4°C to recover the intercellular wash fluid.  The flow-through was pooled for each treatment and concentrated by syringe filtration using a 0.20 µm Micropore Express Plus membrane placed within a removable filtering syringe tip adapter. Filters were placed in homogenization tubes and flash frozen in liquid nitrogen. The filter membranes were homogenized in Trizol Reagent for 1 min at 1750Hz using a GenoGrinder followed by the Direct-Zol Miniprep Plus kits, which included an on-column DNAse I treatment (30 U/column for 15 minutes). RNA samples were additionally treated with TURBO DNase to eliminate DNA contamination. Total RNA underwent RiboZero treatment to remove bacterial rRNA. Illumina TruSeq Stranded Total RNA Sample Prep Kit was used for the construction of sequencing libraries. RNA libraries were prepared for sequencing using standard Illumina protocols