NB: This dataset is currently under embargo while the data is being verified.
For more details, please see my thesis "Investigating the effects of temperature on bacterial pathogens of common bean".
Aim: Determine which genes are transcriptionally affected by temperature and oxidative stress in plant pathogenic Pseudomonas (Pseudomonas savastanoi pv phaseolicola 1302A and Pseudomonas syringae pv syringae B728a)
Materials and Methods: Glycerol stocks of Pspph 1302a and Pss B728a were streaked on LBA plates supplemented with 100 µg/ml nitrofurantoin and left to incubate for 2 days at 28°C. A single colony for each strain was then grown in 10 ml LB in a 50 ml falcon tube overnight at 28°C in a shaking incubator (220 rpm, Innova 4230 refrigerated incubator shaker). The next day, the cell cultures were pelleted down in a centrifuge set at 3000xg rcf at room temperature (22°C) for 3min. The supernatant was discarded, cultures were resuspended in 10ml milliQ. This process was repeated another two times. The pellet was then resuspended in MG medium, and each bacterial culture was adjusted to an OD600 of 0.1. Aliquots of 5ml of these cultures in MG in 50ml falcon tubes were incubated at the temperatures of interest (21 vs 28°C) in a shaking incubator (220rpm, Innova 4230 refrigerated incubator shaker) for 4h so that they were in early log phase. 50µl of 100X concentrated H2O2 stock diluted in milliq or milliQ was added to each culture. The final H2O2 concentrations were: 20mM for 1302a and 80mM for B728a. These were then allowed to incubate for another 30min before 10ml of RNAlater reagent (Invitrogen) was added to stabilise the RNA content. The tubes were then centrifuged at 3000xg rcf for 10min at 4°C. The supernatant was carefully removed and the pellet was flash frozen in liquid nitrogen and stored at -80°C until RNA extraction.
Frozen pellets were resuspended in 600µl lysis buffer RLT plus from the RNeasy plus mini extraction kit (Qiagen) supplemented with 10ul/ml β-mercaptoethanol according to the manufacturer's instructions. This mixture was then vortexed for 1min, and then first loaded through a gDNA column to remove the bulk of the genomic DNA. The runoff was then mixed with 600µl of 70% ethanol and loaded onto a RNA spin column. After centrifuging for 1min at 9000xg rcf on a benchtop centrifuge (Fisher Scientific accuSpin Micro17) the runoff was discarded and the column was treated with 80µl DNAse I in buffer (RNAse-free DNAse kit, Qiagen) according to the manufacturer's specifications. The DNAse treated column was incubated for 20 min at 28°C and then the column was washed as per the manufacturer’s recommendations. Finally, the RNA was eluted from the column with 50µl 37°C ultra pure DEPC-treated water into a fresh nuclease-free eppendorf tube. The eluate was then treated with Turbo DNAse (Turbo DNA-free kit, Invitrogen) according to the company's instructions: 1.5µl Turbo DNAse was added to each sample as well as the required buffer, and samples were incubated at 37°C for 20min, before another 1.5µl DNAse was added and a second incubation period at 37°C for 30min. The DNAse was then inactivated with 0.2 volume of deactivation beads incubated in the samples for 5min and centrifuged at 17000xg rcf, RT. The supernatant was transferred to a new nuclease-free Eppendorf tube. The concentration and purity of the RNA was checked using a Nanodrop spectrophotometer and the Qubit's RNA and DNA HS quantification kit. The levels of DNA and protein contaminants were still too high so the Turbo DNAse treatment was repeated. The RNA was purified from the resulting mixture using the RNeasy plus kit. For this clean-up step, fresh columns were used with an extra washing step with RPE buffer for a total of 3 washes instead of 2. Furthermore, I incubated the samples in RW1 wash buffer for 5min instead of centrifuging straight away. To counter the potential reduction of RNA concentration from this second step, RNA was eluted in a lower volume than in the first round - volume 35µl. RNA was then quantified using the Qubit and Nanodrop, the levels of protein and DNA contamination were also measured by the Nanodrop (ThermoScientific Nanodrop One) and the Qubit (Invitrogen Qubit 4 Fluorometer). Samples were then frozen and stored at -80°C until they were sent for sequencing. As per the sequencing company’s requirements, only samples which had A260/280 and A260/230 ratios above 2, more than 500 ng RNA total and ≥ 6.0 RNA integrity number were sent for sequencing. To check that the DNA had been removed from the samples, a sample that had fit these readings on the nanodrop and Qubit and could have been sent for sequencing was treated with RNAse and then loaded onto an agarose ethidium bromide gel to double check for DNA contamination. No DNA bands were detected in the sample even though 8µl were loaded. This together with the Nanodrop and Qubit readings, confirmed that sample quality was high enough for sequencing.
A total of 34 samples (n=3 replicates per treatment combination) were sent to Novogene on dry ice for sample QC and sequencing: 24 samples and 10 extra substitute samples. Two samples failed the QC check (j9 and j14) and were thus replaced by their corresponding substitute. The 24 samples were then sequenced by the company.
Library preparation and paired-end sequencing 250bp Illumina sequencing was performed by the company according to their company policy: Ribosomal RNA was removed using the Illumina Ribo-Zero Plus rRNA Depletion Kit, followed by ethanol precipitation. The remaining RNA was then fragmented and the first strand cDNA was synthesized using random hexamer primers. During the second strand cDNA synthesis, dUTPs were replaced with dTTPs in the reaction buffer. The sequencing library was made using Novogene’s NGS Stranded RNA Library Prep Set (PT044) which includes end-repair, A-tailing, adapter ligation, size selection USER enzyme digestion, amplification and purification. For this library, the size selection was between 250 and 300bp. Finally, pooled Illumina sequencing was performed on this library.
RNA sequencing data was first processed using kallisto version 0.48.0 (Bray et al., 2016). Details of the code used can be found in github, link to the repository here: https://github.com/JuliannaPiat/Psy_RNAseq_screen. K-mers derived from reads were pseudo-aligned to their genomic sequence (1302a assembly GCF_001294025.1 and B728a assembly GCA_000012245.1 respectively) and the TPM (transcripts per million) was recorded. This quantified data was analysed and graphed using DeSeq2 version 1.36.0 (Love et al., 2014) from Bioconductor 3.15 on R version 4.2.1. DeSeq2 was used to calculate the log2 fold change (L2FC) between treatments and the L2FC was used as the quantitative measure of gene expression change for the rest of the data analysis. DeSeq2 also calculated the corresponding P-value associated with the L2FC, and this P-value adjusted to reduce false discovery rate using the Benjamini-Hochberg Procedure was the P-value used for determining significance. Genome and Proteome annotations from UniProt, NCBI, KEGG and Pseudomonas.com were used to identify transcripts by gene name / function. In the case of Pspph 1302A, annotations from Pspph 1448A were mostly used because 1448A is the reference genome for Pspph in these databases.
