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Sample GSM7312085

Query DataSets for GSM7312085

Status

Public on Sep 27, 2023

Title

bphOP1_KO_blue, rep 2

Sample type

SRA

Source name

bacteria

Organism

Pseudomonas syringae pv. syringae B728a

Characteristics

strain: B728a
cell type: bacteria
genotype: delta_bphOP1
treatment: blue

Treatment protocol

Two-milliliter culture aliquots were transferred into individual wells of replicate 6-well cell culture plates and plates were immediately exposed to blue light (20 μmol/m^2/s), red light (30 μmol/m^2/s), far-red light (188 μmol/m^2/s), or white light (34 μmol/m^2/s), or maintained in the dark for 15 min at 20–22°C without shaking. As a follow-up to prior microarray studies, the Δlov mutant was subject to two additional treatments in which cells cultivated in defined HMM medium were exposed for 15 min to blue light in the presence or absence of 0.23 M NaCl.

Growth protocol

Two independent cultures of each strain were grown on each of six separate days. For each culture, an initial culture was grown from a single colony in KB medium containing Rif (50 μg/mL) to a density of approx. 10^9 cells/mL, at which point 10 μL was sub-cultured into 5 mL KB medium and grown again to a density of approx. 10^9 cells/mL. A 0.2-mL aliquot was sub-cultured into 20 mL KB in a 125-mL flask and grown for 4–6 h with shaking in the dark at 20–22°C until an optical density at 600 nm (OD600) of approx. 0.5.

Extracted molecule

total RNA

Extraction protocol

Immediately after treatment, 0.5 mL of each culture sample was added to 1 mL of RNAprotect (Qiagen) and vortexed for 5 sec. Following a 5-min incubation at room temperature, the cells from the two independent cultures for a single strain x treatment combination were combined and centrifuged at 5,000 x g for 10 min. The supernatant was decanted, and the cell pellet was stored at -70°C. For each strain, this process was repeated on six separate days. The cell pellets for a given strain x treatment combination collected on two separate days were thawed on ice and combined, and this pool was considered one biological replicate. In this manner, each strain x treatment combination was represented by three biological replicates, each of which contained cells derived from four independently exposed cultures. Total RNA was extracted using a RNeasy Mini Kit (Qiagen) with on-column Dnase I treatment, with one biological replicate for each strain x treatment combination extracted at the same time.
Library construction and sequencing were performed by BGI Genomics (China). For each sample, total RNA was depleted for ribosomal RNA using an Epicentre Ribo-Zero Magnetic Kit (Bacteria), fragmented, and then used for RNA-seq library construction using a TruSeq RNA Sample Prep Kit v2 (Illumina). Libraries were assessed for quality using a 2100 Bioanalyzer, quantified using real-time quantitative PCR, and sequenced on an Illumina HiSeq 4000 with 100-bp paired-end reads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

total RNA

Data processing

The raw reads were filtered by BGI Genomics using SOAPnuke (v1.5.2) to remove reads containing sequencing adapters and low-quality reads (reads containing >20% of bases having quality scores ≤15 or an ambiguous sequence content of >1%). FASTQ files of the resulting “clean” reads were used for further quality assessment using FastQC v0.11.4 and analysis.
The clean reads from each sample were mapped to the B728a reference genome GCF_000012245.1 (2005 version) using Bowtie2 (v2.2.6) and TopHat2 (v2.1.0).
The number of reads that mapped to each gene in the genome were counted using HTSeq-count (v0.6.0) with parameters -i locus_tag -t gene -s reverse -r pos -m intersection-nonempty -a 10 -f bam. Genes with average read counts of <1 across all samples were excluded from subsequent analyses.
Counts were normalized for library size using upper-quartile normatilzation.
Normalized gene counts for all 66 samples were used to assess differential expression between specified strain x treatment combinations using the R package QuasiSeq (negative binomial model, strain x treatment interaction and replicate used as fixed effects, and the QLSpline method for FDR estimates), with q-value ≤ 0.00105 used to define statistical significance.
Assembly: Pseudomonas syringae B278a, GCF_000012245.1 assembly was downloaded from NCBI in October 2016; the original RefSeq annotation (submitted in 2005 by the Joint Genome Institute) was replaced replaced by a PGAP annotation in 2023 without changing the accession number, but the original annotation used here is still available under NCBI accession GCA_000012245.1
Supplementary files format and content: raw_counts.csv is is a matrix table with raw gene counts for every gene and every sample
Supplementary files format and content: norm_counts.csv is a matrix table with natural log-transformed normalized gene counts for all genes with average raw read counts of ≥1 across all samples
Supplementary files format and content: B728aLight_QuasiSeq_results.xlsx is an Excel file with the results of the QuasiSeq differential analysis results (Ln[fold change], Log2[fold change], fold change, q-value) for all genes along with transcript metadata including gene name, product description, and functional classification.

Submission date

May 09, 2023

Last update date

Sep 27, 2023

Contact name

Gwyn A Beattie

Organization name

Iowa State University

Department

Plant Pathology, Entomology, & Microbiology

Street address

2213 Pammel Dr