title: Illumina HiSeq 2000 sequencing; Leaf transcriptomes of Arabidopsis thaliana colonized by representative phyllosphere commensals alone and in combination with the pathogen Pseudomonas syringae DC3000
design: Leaf transcriptomes of Arabidopsis thaliana colonized by representative phyllosphere commensals alone and in combination with the pathogen Pseudomonas syringae DC3000
library_construction_protocol: Arabidopsis thaliana Col-0 were grown in a gnotobiotic microbox system as described in Innerebner et al. 2011. Briefly, microboxes containing 70 ml MS medium including vitamins (Duchefa) supplemented with 3% sucrose and 0.55% plant agar (Duchefa) were prepared. The medium was covered with a sterile gas-permeable foil (lumox film 25, Sarstedt) containing 8 holes (4 mm diameter) and boxes were UV-irradiated for 20 min. Seeds were surface sterilized and placed on the agar surface at the positions of the 8 holes. Seeds were inoculated with as described in Innerebner et al. 2011. Plants were grown in a growth chamber set to 22°C with 16 h light/8 h dark cycle for the first week followed by 9h light/15 h dark cycle.  "One day before infection with Pseudomonas syringae pv. tomato DC3000 luxCDABE (Fan et al. 2008, The Plant Journal 53:393-399), 30 holes were pierced into the microbox walls with a canula (BIBRAUN, 18G x 1.5\"", ø 1.2 x 40 mm) to reduce relative humidity to roughly 80%. One loop of a two-day Pst lux culture on King's B (KB) plate was suspended in 1 ml 10 mM MgCl2. 150 μl were plated onto a KB plate and incubated overnight at 28°C to grow the infection inoculum. For preparation of the infection suspension, 10 ml 10 mM MgCl2 were spread on the plate and after 10 minutes the suspension was transferred to a 50 ml falcon tube. The concentration was adjusted turbidimetrically to an OD600 of 0.1 and further diluted 1:100 to an OD600 of 0.001 which corresponded to approximately 5x105 cfu ml-1. Three-week old plants were kept in darkness before infection. Plants were infected by spraying approximately 100 μl of Pst lux suspension over each microbox (four times spraying in total) under sterile conditions. As a control, uninfected plants were sprayed with 10 mM MgCl2." Four independent experiments were conducted to collect plant material for transcriptome analysis, with each corresponding to one biological replicate. Leaves for RNA extraction were collected at midday. For each treatment, single leaves were removed from 18 plants (out of three microboxes), directly frozen in liquid nitrogen and then stored at  80°C until RNA extraction. Total RNA was extracted from 18 pooled leaves using the RNeasy mini Plant kit (Qiagen) including the on-column DNaseI digest according to the manufacturer's recommendations.  The quality of the isolated RNA was determined with a Qubit® (1.0) Fluorometer (Life Technologies) and a Bioanalyzer 2100 (Agilent). Libraries were prepared using the TruSeq RNA Sample Prep Kit v2 (Illumina). Briefly, total RNA samples were polyA-enriched and then reverse-transcribed into double-stranded cDNA. The cDNA samples were fragmented, end-repaired and polyadenylated before ligation of TruSeq adapters containing the index for multiplexing. Fragments containing TruSeq adapters on both ends were selectively enriched with PCR. The quality and quantity of the enriched libraries were validated using Qubit® (1.0) Fluorometer and the Caliper GX LabChip® GX and had an average fragment size of approximately 260 bp. The libraries were adjusted to 10 nM in Tris-Cl 10 mM, pH 8.5 with 0.1% Tween 20. The TruSeq SR Cluster Kit v3-cBot-HS (Illumina) was used for cluster generation using 10 pM of pooled normalized libraries on the cBOT.