title: GSM9661609: wildtype, flg22 Rep2; Pseudomonas syringae pv. tomato str. DC3000; RNA-Seq
library_name: GSM9661609
library_construction_protocol: At 5 hpi, leaves from each treatment were harvested by cutting at the leaf blade junction. Briefly, 80-100 leaves were arranged on four sheets of parafilm, rolled and inserted into the barrels of four 20-ml syringes. An ice-cold RNA stabilizing buffer (Invitrogen) was poured into each syringe, which was sealed and vacuum infiltrated at 95 kPa for 2 min, followed by a slow release of the vacuum. The RNA stabilizing buffer was spun out from apoplast at 1000× g for 10 min at 4°C to isolate bacterial RNA. The flow-through was pooled for each biological replicate form each treatment and was concentrated by syringe filtration, using a 0.20-μm Micropore membrane (Millipore). Filters were placed in homogenization tubes, were flash frozen in liquid nitrogen, and were stored at −80°C for RNA extraction.The filter membranes were homogenized in Trizol reagent (Thermo Fisher Scientific, Waltham, MA) for 1 min at 1,750 Hz, using a Geno/Grinder (Thermo Fisher Scientific, Waltham, MA) followed by the chloroform-ethanol isolation from manufacture (TRIzol™ Reagent User Guide, Pub. No. MAN0001271 D). For in vitro samples, 1 ml of the 1 × 109 CFU/ml bacterial inoculum in 0.25 mM MgCl2 was pelleted and RNA was extracted followed by the same procedure above. The plant samples were depleted of rRNA using the QIAseq FastSelect –rRNA Plant Kit (Qiagen, Germantown, MD) and bacteria samples were depleted of rRNA using the QIAGEN FastSelect rRNA HMR Kit.
identifiers.namespace: GEO
identifiers.id: GSM9661609_r1