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Sample GSM1595233

Query DataSets for GSM1595233

Status

Public on Feb 01, 2015

Title

wild type, after Fe(II) shock, replicate 2

Sample type

SRA

Source name

planktonic cells

Organism

Pseudomonas aeruginosa

Characteristics

strain: UCBPP-PA14
genotype: WT-pMQ72
growth phase: late stationary
length fe(ii) shock: 30 min
fe(ii) concentration: 200 µM ferrous ammonium sulfate
medium: 100 µg ml-1 gentamycin, 1% arabinose, 40 mM succinate (C4H4Na2O4 · 6H2O), 9.3 mM NH4Cl, 2.2 mM KH2PO4, 25 mM KNO3, 25 mM NaNO3, 25 mM MOPS, 25 mM NaMOPS pH 7.2. Additionally, immediately prior to inoculation 100 µM CaCl2, 1 µM (NH4)2Fe(SO4)2 · 6H2O, 1 mM MgSO4, and trace metals (Kopf SH, Henny C, Newman DK. 2013. Ligand-enhanced abiotic iron oxidation and the effects of chemical versus biological iron cycling in anoxic environments. Environ Sci Technol 47:2602-2611) were added

Treatment protocol

when the cells reached deep stationary phase (OD500 = 0.8), 1 ml of culture was added to 2 ml RNAprotect before and after a 30 min 200 µM (NH4)2Fe(SO4)2 · 6H2O shock. RNAprotect treated cells were pelleted by centrifugation for 10 min. at 5000 x g. the pellets were frozen and stored at -80 degrees C until RNA extraction.

Growth protocol

Triplicate aerobic cultures of P. aeruginosa WT-pMQ72 and ∆bqsR-pMQ72 were grown in MOMM + 100 µg ml-1 gentamycin at 37 oC for 36 hours. Anaerobic cultures were inoculated with 1% inoculum in MOMM + 100 µg ml-1 gentamycin + 1% arabinose. when the cells reached deep stationary phase (OD500 = 0.8).

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted using the RNEasy kit (Qiagen) according to the manufacturer's instructions with minor modifications. Briefly, frozen pellets were resuspeded in 215 ul TE buffer containing 15 mg/ml lysozyme (Sigma) and 1.4 mg/ml proteinase K (Qiagen). Samples were incubated with shaking for 10 minutes at room temperature to disrupt the cell walls. 700 ul buffer RLT and 500 ul 100% ethanol were added and the mixture was applied to the Qiagen column. washing, and elution were carried out according to instructions in 100 µl water. Total RNA was then further treated with the rigoruos treatment Turbo DNAse free kit (Ambion) according to instructions. RNA was concentrated with an ethanol precipitation and 5 ug total RNA was subjected to ribosomal RNA depletion using the Ribo-Zero Magnetic Kit for gram negative bacteria (Epicentre).
Libraries for RNA Seq were constructed from the rRNA-depleted samples using the NEBNext mRNA Library Prep Master Mix Set for Illumina, according to the manufacturer's instructions.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Base calling, de-multiplexing, and conversion of sequencing to fastq format were carried out by Illumina software on the Hi Seq 2500 machine.
Reads were trimmed and filtered based on their quality scores using the Trimmomatic package v 0.32 (Bolger et al, Bioinformatics published online April1 2014). Leading and trailing quality cutoffs were set to 27, and a cutoff of 20 was set for a sliding window of 4 bases. Reads shorter than 35 bp after trimming were discarded.
The trimmed reads were mapped to the genome using Bowtie 1.0.1 (Langmead B, Trapnell C, Pop M, Salzberg S. 2009. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biology 10:R25.) and sorted mapped reads to indexed .sam file and converted to .bam file with .Samtools v. 0.1.19 (Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, Subgroup GPDP. 2009. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25:2078-2079.),
Read counts per gene or transcriptional unit were calculated using Easy RNASeq package (Delhomme N, Padioleau I, Furlong EE, Steinmetz LM. 2012. easyRNASeq: a bioconductor package for processing RNA-Seq data. Bioinformatics 28:2532-2533.) in R, using .gff gene description files generated from the curated genome hosted by NCBI (NC_008463.1) and .gff transcriptional units files modifying the curated genome hosted by NCBI (NC_008463.1) by the results of the single nucleotide resolution sequencing study published by Wurtzel et al (Wurtzel O, Yoder-Himes DR, Han K, Dandekar AA, Edelheit S, Greenberg EP, Sorek R, Lory S. 2012. The single-nucleotide resolution transcriptome of Pseudomonas aeruginosa grown in body temperature. PLoS Pathog 8:e1002945.)
Normalization, ratios, significance were determined using Degust web-interface (Law C, Chen Y, Shi W, Smyth G. 2014. voom: precision weights unlock linear model analysis tools for RNA-seq read counts. Genome Biol 15:R29.)
Genome_build: gi|116048575|ref|NC_008463.1|
Supplementary_files_format_and_content: 2 mapping schemes: gene mapping and transcriptional unit mapping. The files will be in .csv format which contains average log2 fold change values normalized to the WT before shock condition for all samples/conditions, as well as the raw counts for all 12 samples.
150126_FDR_1_all_conditions_normalized_wt_0_txn_unit_analysis.txt: abundance count, calculated fold change
150126_FDR_1_all_conditions_normalized_wt_0_gene_analysis.txt: abundance count, calculated fold change
BqsR_regulon_txn_unit.txt: Transcription units in Fe(II) shocked cells that wt was over 2 fold change greater or less than ∆bqsR with an FDR < 0.01
BqsR_regulon_genes.txt: Genes in Fe(II) shocked cells that wt was over 2 fold change greater or less than ∆bqsR with an FDR < 0.01

Submission date

Jan 28, 2015

Last update date

May 15, 2019

Contact name

Dianne K Newman

E-mail(s)

dkn@caltech.edu

Organization name

Caltech

Department

BBE and GPS