GSE195976
RpoN is required for a functional type III secretion system in Yersinia pseudotuberculosis
One milliliter of triplicate bacterial cultures in both Wild type (WT) and rpoN mutant were immediately pelleted by centrifugation at 9000 rpm at room temperature for 2 minutes after adding phenol:ethanol. The supernatant were removed and the pellets resuspended in 0.5 ml Trizol solution. For Gram-negative bacteria, the cells were homogenized in Trizol solution by pipetting up and down 15 times. For Gram-positive bacteria, culture suspensions in Trizol were transferred to previously cooled bead beater tubes containing 0.1-mm glass beads and treated with Mini-Beadbeater (Biospec Products Inc, USA) twice at a fixed speed for 45 seconds, and then cooled on ice for 1 minute between the treatments. Culture homogenates were incubated at room temperature for 5 minutes and then supplemented with 0.2 ml chloroform, thoroughly mixed by shaking 10 times, and incubated for 3 minutes. After centrifugation at 12 000 g at 4°C for 15 minutes, the aqueous upper phase was carefully transferred to new RNase free tubes. An equal volume of 99% ethanol was added to the transferred aqueous phase and isolation continued using the Direct-Zol RNA Miniprep Plus (Zymo Research, USA) RNA purification kit protocol. Total RNAs were eluted in RNase free water in RNase free tubes. The total RNA concentrations were measured using the Qubit BR RNA Assay Kit (ThermoFisher Scientific, USA) and RNA integrity confirmed on a 0.8% agarose gel in TBE buffer. All rRNA-depleted RNA-seq library preparations were performed according to Shishkin et al.,2015 with minor modifications. RNAtag-Seq allows multiple library preparations in one tube, with initial tagging of total RNA samples with modified (5'P and 3'ddC) DNA barcoded adaptors, each harboring a unique 8 nt sequence used to demultiplex individual libraries after sequencing We combined the library preparation of three biological replicates from WT and ?rpon preparations in one tube for each bacterial strain. Therefore, 24 unique barcoded adaptors were used to tag the 24 replicates. A total of 100 ng total RNA was used for each biological replicate. The total RNA was fragmented in 2X FastAP Thermosensitive Alkaline Phosphatase buffer for 3 minutes at 94°C, DNase treated, and dephosphorylated with a combination of TURBO DNase and Thermosensitive Alkaline Phosphatase in 1X FastAP buffer for 30 minutes at 37°C. Fragmented, DNase-treated, and dephosphorylated total RNAs were cleaned with a 2X reaction volume of Agencourt RNAClean XP beads. Cleaned total RNAs were incubated with 100 pmol of a unique DNA barcode adaptor at 70°C for 3 minutes, and then ligated with T4 RNA ligase 1 for 90 minutes at 22°C. The ligation was stopped and the enzyme denatured by the addition of RLT buffer. The 36 denatured ligation mixes were then pooled and cleaned in a Zymo Clean & ConcentratorTM-5 column according to the manufacturer's 200 nt cut-off protocol. The RNAs were eluted in 32 ml of RNase free water. The ribosomal RNA was depleted using the Ribo-ZeroTM Magnetic Gold Kit (Bacteria) according to the manufacturer's instructions. The first strand cDNA of each pool was generated using an AffinityScript Multiple Temperature cDNA synthesis kit with 50 pmol of AR2 primer at 55°C for 55 minutes. The RNA was degraded by adding a 10% reaction volume of 1 N NaOH at 70°C for 12 minutes and the reaction neutralized with an 18% reaction volume of 0.5 M acetic acid. After cleaning the reverse transcription primers with a 2X reaction volume of RNAClean XP beads, the 3Tr3 adapter was ligated with T4 RNA ligase 1 at 22°C with overnight incubation. The second ligation was cleaned first with a 2X, and secondly 1.5X, reaction volume of RNAClean XP beads. The cDNA was then used as the template for PCR reaction with FailSafeTM PCR enzyme mix using 12.5 pmol 2P_univP5 as forward primers and 12.5 pmol ScriptSeqTM Index (barcode) PCR primers as reverse primers. The PCR cycles were as follows: 95°C for 3 minutes, followed by 12 cycles at 95°C for 30 seconds, 55°C for 30 seconds, and 68°C for 3 minutes, and then finishing at 68°C for 7 minutes. The PCR product was cleaned first with a 1.5X, and secondly 0.8X, reaction volume of AMPure beads and eluted in RNAse free water. The library concentrations were measured using the QubitTM dsDNA HS Assay Kit and the library insert size determined by the Agilent DNA 1000 Kit in a 2100 Electrophoresis Bioanalyser Instrument (Agilent, USA). The libraries were sequenced by Illumina NovaSeq 6000 Overall design: Transcriptomics