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Status |
Public on Jan 09, 2021 |
Title |
G. diazotrophicus cells, 37 µM FeCl3 R3 |
Sample type |
SRA |
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Source name |
Cultured cells
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Organism |
Gluconacetobacter diazotrophicus |
Characteristics |
strain: PAL5 treatment: 37 µM FeCl3
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Treatment protocol |
Subsequently, cultures were split into aliquots of 25 ml, put into 250 ml flasks and treated with FeCl3 (37 µM) or left untreated. This was done to ensure that all cells would be in the same growth phase for transcriptome sequencing (RNA-seq) analysis. After 30 min of incubation, cells were collected (5,000 rpm, 10 min, 4°C) and stored at -70°C.
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Growth protocol |
PAL5T strain were cultured in 5 ml of modified LGI-P medium at 30°C and 180 rpm for approximately 48 hours. After this period, a pre-inoculum (1.0 × 106 cells) was suspended in 100 µl of saline solution [0.7 % NaCl (w/v)] and inoculated in flasks containing 250 ml of modified LGI-P medium without the addition of FeCl3. The erlenmeyer flask was then incubated at 30°C and 180 rpm until the cell density reached the exponential growth phase (0.6 O.D.600 ml-1) (Optic density at wavelength 600 nm).
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was isolated with Trizol in accordance with the manufacturer’s protocol (Life Technologies) and treated with DNase I (Epicenter) in order to remove genomic DNA contamination. RNA purity was quantified using Qubit (Life Technologies). Seven micrograms of total RNA were used to ribosomal RNA (rRNA) depletion with MICROBExpressTM kit. The efficiency of depletion was evaluated in agarose gel electrophoresis (1%) followed by quantification of the total RNA with Agilent 2100 Bioanalyzer (Agilent). A total of 500 ng mRNA was used for the contruction of a sequencing library using the standard protocol of the SOLid Total RNAseq Kit (Life Technologies). The libraries were barcoded by using the SOLiD Transcriptome Multiplexing Kit (Life Technologies). The emulsion PCR and sequencing were performed according to Ion One TouchTM 200 Template Kit v2 DL and Ion PITM Sequencing 200 Kit v2 using the standard Life Technologies protocols, respectively. The Ion Proton Semiconductor Sequence (Life) was used to sequence twelve libraries generated from three biological replicates from each independent treatment.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Ion Torrent PGM |
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Data processing |
CLC Genomics Workbench version 7.5.1 program Reads containing ≤ 20 bp, and those mapping to rRNA 5S, 16S and 23S genes, were all discarded. The following parameters were used: the reads were trimmed for 20 bp, minimum 90% alignment with the reference sequence and 80% identity for inclusion as mapped read and the number of hits equal 1. The genes were considered expressed if more than three times of coverage was present. They were considered to be differentially expressed genes when the fold change was bigger than 1.2 or smaller than -1.2 and the p-value was smaller than 0.05 by t’test. Gene ontology analysis was performed using the bioinformatics tool Blast2GO (https://www.blast2go.com/) (70). The genes were function classified by COG (Clusters if Orthologous Groups of proteins, (http://www.ncbi.nlm.nih.gov/COG). Real-time quantitative (RT-qPCR) was used to validate the transcriptome. Genome_build: genome of Gluconacetobacter diazotrophicus PAL5 (NCBI accession number NC_010125.1) Supplementary_files_format_and_content: Tab delimited text files include the reads of each biological sample, RPKM values for each Sample and statistical analysis.
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Submission date |
Jan 08, 2021 |
Last update date |
Jan 09, 2021 |
Contact name |
Leonardo Araujo Terra |
E-mail(s) |
leonardoterra@hotmail.com.br
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Organization name |
Embrapa Agrobiologia
|
Lab |
Genetics and biochemistry laboratory
|
Street address |
Rodovia BR-465
|
City |
Seropédica |
State/province |
RJ |
ZIP/Postal code |
23897-970 |
Country |
Brazil |
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Platform ID |
GPL29580 |
Series (1) |
GSE164445 |
Transcriptomic Response of the Diazotrophic Bacteria Gluconacetobacter diazotrophicus Strain PAL5 to Iron Limitation and Characterization of the fur Regulatory Network |
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Relations |
BioSample |
SAMN17258053 |
SRA |
SRX9806236 |