title: Illumina HiSeq 2000 paired end sequencing; RNA-seq analysis of Pseudomonas syringae pv. tomato DC3000
design: RNA-seq analysis of Pseudomonas syringae pv. tomato DC3000
library_name: Pto+Naringenin_p
library_construction_protocol: Pto DMSO, Pto +Ft and Pto +Na cells were harvested at OD660=1.5, washed with sarkosyl 0.1% and cell pellets were immediately frozen in liquid nitrogen and conserved at -80ºC until RNA isolation. Pseudomonas syringae pv. tomato (Pto) DC3000 was grown in LB at 28ºC to an OD660=0.5 in the absence and in the presence of phloretin and naringenin.Bacteria were grown on LB plates for 48 hours and resuspended in 3 ml of 10 mM MgCl2. Ten ml of LB with 0.01% DMSO, 0.5 mM phloretin or 0.5 mM naringenin (both in DMSO) were inoculated and adjusted to an OD660 of 0.05. The strains were grown at 28ºC to an OD660=0.5. rRNA removal with Ribo zero magnetic kit was performed by Macrogen. The cells were processed for RNA isolation using TRI Reagent®LS (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer instructions, except that the TRI Reagent was preheated at 70ºC and the lysis step was carried out at 65ºC. The RNA obtained was treated with RNase-free DNase I (Quiagen) followed by purification with RNeasy mini kit (Quiagen). RNA concentration was determined spectrophotometrically and its integrity was assessed by agarose gel electrophoresis. cDNA libraries were constructed by Macrogen
attributes.tag: Experimental Factor: compound
attributes.value: naringenin
attributes.tag: Experimental Factor: dose
attributes.value: 0.5