title: hrpX_mutant_3
design: Bacterial strains and growth conditions  The wild-type X. citri subsp. citri (da Silva et al. 2002), and hrpX mutant strains used in this study were described in our previous study (Guo et al. 2011). Both the strains were grown at 28°C in nutrient broth (NB), on nutrient agar (NA), or in NYG medium (Daniels et al. 1984). Antibiotics rifamycin and kanamycin were added to the media at 50 µg/ml final concentrations.  RNA extraction  Total RNA was extracted in triplicates from wild-type and mutant strains as described in our previous study (Guo et al. 2011).  Briefly, strains from NA plates were grown in NB medium at 28ºC until mid-exponential phase. Cultures were harvested by centrifugation and inoculated in to nutrient-deficient XVM2 medium, after washing the pellet once with the same medium. Cultures were finally harvested for RNA extraction, when the optical density at 600 nm reached the value of 0.4, and mixed immediately with RNAprotect bacterial reagent (Qiagen, Valencia, CA, and U.S.A.). Total RNA was extracted from each replicate separately using RiboPure bacteria kit (Ambion, Austin, TX, USA), according to manufacturer's instructions. Genomic DNA contamination from extracted RNA samples was removed using TURBO DNA-free kit (Ambion). Amount and the quality of the RNA samples was initially determined using NanoDrop™ 1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE). Samples with absorbency at 260/280 and 260/230 nm ratios > 2 were subjected to further processing.  mRNA enrichment and RNA-seq(uencing)  Total RNA samples were enriched for mRNA, by depleting rRNA using MICROBExpress kit from Ambion following the manufacturer’s instructions. Enriched samples were checked for integrity using Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).  RNA samples that passed the quality control were sequenced using the Illumina Genome Analyzer IIx (GAIIx) system by following the standard protocol at the Center for Genome Analysis at Yale University. Real-time analysis and base calling were performed using the CASAVA v1.6 pipeline.
library_name: hrp_mutant_3