GSM4777789: P. syringae B728a filter rep 1; Pseudomonas syringae pv. syringae B728a; RNA-Seq
Cells in broth cultures were harvested by pipetting 1 ml of suspension into a 15 mL conical tube containing 125 µl ice-cold EtOH/Phenol stop solution (5% water-saturated phenol (pH<7.0) in ethanol). Filters were immersed in the EtOH/Phenol stop solution and cells were harvested by sonication for 30 seconds followed by vortexing for 20 seconds to ensure complete cell detachment from the filters. Cells were then collected by centrifugation at 12,000 rpm (13,800 x g) for five minutes at 4°C. Supernatant was decanted and the cells were frozen in liquid nitrogen and stored at -80℃ until RNA isolation. RNA isolation was performed using a Direct-zol™ RNA Kit (Zymo Research). RNA was stored frozen at -80°C. 1 µl of each sample was diluted into 4 µl of RNase free water and submitted to the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley where Ribo-Zero was used for rRNA removal. RNA abundance and purity were determined using a 2100 Bioanalyzer (Agilent Technologies) and quantified using Qubit (Invitrogen). After reverse transcription, shearing of cDNA, size fractionation, and Illumina library production, the Vincent J. Coates Genomics Sequencing Laboratory samples were sequenced using an Illumina HiSeq4000 platform with 50 base pair, single-end reads. Three biological replicates were sequenced per treatment.