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| Status |
Public on Mar 07, 2022 |
| Title |
6 h, P. putida + nZVI, 3 |
| Sample type |
SRA |
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| Source name |
bacterial pellet exposed to nZVI
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| Organism |
Pseudomonas putida |
| Characteristics |
strain: NCTC 10936
treatment: exposed to 100 mg/L nZVI
time: 6 h
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| Treatment protocol |
Natural reservoir water from a local reservoir in Czech Republic (50.7702097N, 15.0755733E) was collected to a depth of 10 cm from a surface point in a spade on November 18, 2018 and was used as the NP exposure medium in the current study. P. putida from its mid-log phase was pelleted down and added into the reservoir water, and exposed to 100 mg/L nZVI or 44.5 µg/L dissolved iron), a flask without nanoparticle was served as control condition
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| Growth protocol |
P. putida was grown in 10 mL tryptone soy medium (7 h at 30°C, 150 rpm), to obtain the mid-log exponential growth phase of cells (approximately 3.3 × 10^8 cells/mL). The cells were subsequently harvested, pelleted down, and transferred to reservoir water as an exposure medium for the nZVI toxicity studies.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Samples treated with nZVI particles were removed by a KV-70-70-25-N dimensional nickel-plated block magnet (UNIMAGNET, Czech Republic) before RNA extraction. The RNA was harvested after 0, 3, 6, and 24 h from nZVI-exposed cells and after 0 and 24 h from dissolved iron-exposed cells using an Isolate II RNA Mini Kit (Bioline, Canada). Cell pellets were obtained through centrifugation at 21,000 × g, 4°C for 15 min and added immediately 1 mg/mL of lysozyme and incubated at 37°C for 10 min prior to the addition of RNA lysis buffer. The subsequent DNase treatments and RNA washing steps were then treated as described in the manufacturer's protocol. The integrity of the total RNA (RIN) was determined with an Agilent RNA ScreenTape by 2200 TapeStation system (Agilent Technologies, Germany) and Qubit™ RNA Broad Range Assay Kit using Qubit 2.0 fluorometer (Life Technologies, USA) was for RNA concentration quantification.
To prepare for whole transcriptome sequencing of the RNA-seq library, triplicate biological flasks containing starting material with at least 700 ng of total RNA (RIN > 7) were depleted of rRNA using a NEBNext rRNA depletion kit for bacteria (New England Biolabs, USA) following the manufacturer’s instructions. Then, the RNA was prepared using a NEBNext® Ultra™ II RNA Library Prep kit for Illumina® (New England Biolabs). The resulting cDNA libraries were pooled and were sequenced using an Illumina NextSeq system with an 85-bases read length in the forward direction
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
6hr_nZVI_3
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| Data processing |
The first 15 nucleotides of the reads of the adapters were trimmed, and the adapters were aligned to the P. putida KT2440 reference genome (GenBank Accession no. AE015451.2) using STAR aligner and assessed for transcript level (ht-seq count).
The differential gene expression for each condition was illustrated using the relative log expression function in the DESEQ2 package, and statistical analysis was performed through the p.adjust function in R software.
Genes showing log2 (fold change) of ≥1.0 or ≤ −1.0 and adjusted P-value < 0.05 were considered to be significantly expressed. Clustering and visualization of the dissimilarity matrix was achieved using pheatmap. PCA was performed based on the PCAtools package
Genome_build: Pseudomonas putida KT2440
Supplementary_files_format_and_content: gene-clustering-genes-with-highest-variance.xlsx
Supplementary_files_format_and_content: most-changed-genes1.xlsx
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| Submission date |
Mar 04, 2022 |
| Last update date |
Mar 07, 2022 |
| Contact name |
Alena Sevcu |
| E-mail(s) |
alena.sevcu@tul.cz
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| Phone |
+420777341820
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| Organization name |
Technical University of Liberec
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| Department |
Applied Biology
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| Street address |
Bendlova 1409/7
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| City |
Liberec |
| State/province |
Liberec |
| ZIP/Postal code |
46117 |
| Country |
Czech Republic |
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| Platform ID |
GPL22319 |
| Series (1) |
| GSE197899 |
Dissolved iron released from nanoscale zero-valent iron (nZVI) activates the defense system in bacterium Pseudomonas putida, leading to high tolerance to oxidative stress |
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| Relations |
| BioSample |
SAMN26429535 |
| SRA |
SRX14365216 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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