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| Status |
Public on Oct 10, 2024 |
| Title |
SA_15h_1 |
| Sample type |
SRA |
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| Source name |
14028S
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| Organism |
Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S |
| Characteristics |
strain: 14028S
treatment: Salmonella-Acanthamoeb coculture 15h
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| Treatment protocol |
‘Rich medium’: bacterial cells were grown overnight in 10 mL LB, diluted 1:1000 into 10 mL LB, grown at 190 rpm and 37 °C in a 50 mL tubes until the middle of the exponential growth phase (A600 = 0.4). ‘Poor medium’: bacterial cells were grown overnight in 10 mL LB, diluted 1:1000 into 10 mL LB, grown at 190 rpm and 37°C in a 50 mL tubes until the middle of the exponential growth phase (A600 = 0.4). Then bacteria were washed thrice in sterile PAS by centrifugation at 1200× g for 5 min at 25 °C and were grown for 2 h at 25 °C in sterile PAS. ‘Peroxide shock’: bacterial cells were grown overnight in 10 mL LB, diluted 1:1000 into 10 mL LB, grown at 190 rpm and 37 °C in a 50 mL tubes until the middle of the exponential growth phase (A600 = 0.4) then H2O2 in the final concentration of 1 mM H2O2 for 10 min were added. Amoeba axenic culture was grown for 7 days in PYG medium to cell density 4-5×105 cells in 1 ml. Cells were collected by centrifugation at 800×g, washed twice with sterile Page’s Amoeba Saline (PAS) (Page, 1976) and suspended in 10 ml of fresh PAS medium. Salmonella were grown in LB broth at 37 °C to early stationary growth phase (A600 = 2.0). Cells were collected by centrifugation at 3000×g for 5 minutes, washed twice with PAS, then were added to Acanthamoeba at a bacteria:amoeba ratio of 100:1. After 1 hour of infection, extracellular bacteria were killed by adding media with gentamicin in final concentration 100 μg mL−1 and incubated for 1h at 25°C. The medium was then changed to ‘maintenance media’ containing 10 μg mL−1 gentamicin for the rest of the experiment.
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| Growth protocol |
Salmonella was grown in Luria broth at 37°C without antibiotics. A. castellanii axenic culture was grown for 5 d in PYG medium to a density of 4-5 × 105 cells/mL.
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| Extracted molecule |
total RNA |
| Extraction protocol |
The Salmonella were collected by centrifugation. Total RNA was extracted using RNA Extract Reagent (Evrogen, Russia) according to the manufacturer's protocol. DNA contaminants were removed using RNase-free DNase I kit (Ambion, USA).
The integrity of the RNA was checked by Qsep100 bioanalyzer (BiOptic Inc.,Taiwan). Total RNA from Salmonella (5 μg) or Salmonella plus Acanthamoeba (10 μg) was capped with 3´ desthiobiotin-GTP using the Vaccinia Capping System (New England Biolabs, Ipswich, MA, USA). Capped RNA was then purified by Zymo Research Clean and Concentrator™-5 column using manufacturer instructions (Zymo Research, Irvine, CA, USA). Desthiobiotin-GTP-capped RNA was fragmented by polynucleotide kinase buffer and incubated for 5 min at 94 °C. The 3′-phosphates were removed from the fragmented RNA with polynucleotide kinase buffer and T4 polynucleotide kinase at 37 °C for 15 min. Streptavidin enrichment was performed by addition of capped RNA to 30 μL of prewashed hydrophilic streptavidin magnetic beads (New England Biolabs, Ipswich, MA, USA). The biotin-eluted RNA was collected by placing the tube on the magnetic rack. Eluted RNA was cleaned with AMPure XP beads (Beckman Coulter, Brea, CA, USA). Libraries were prepared from capped RNA using the NEBNext small RNA library prep kit (New England Biolabs, Ipswich, MA, USA) according to the manufacturer instructions. Library quality was assessed using a DNA high sensitivity chip on a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) prior to sequencing.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
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| Description |
Salmonella were grown in PAS medium (with 10 μg mL^−1 gentamicin) at 25 °C for 15h.
SA15_1
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| Data processing |
Demultiplexed reads were trimmed against adapters with bbduk (https://sourceforge.net/projects/bbmap/)
Reads were mapped to the A. castellanii genome (GenBank accession number GCA_000313135) and then mapped against the S. Typhimurium 14028s genome (GCA_000022165.1) (Jarvik et al., 2010) using STAR aligner v2.7.11a (Dobin et al., 2013) (https://github.com/alexdobin/STAR) with ‘--outFilterMultimapNmax 1’, ‘--sjdbGTFfeatureExon gene’, ‘--sjdbGTFtagExonParentTranscript gene_id’, ‘--sjdbGTFtagExonParentGene old_locus_tag’, ‘ --quantMode GeneCounts’.
The featureCounts v1.4.6-p5 function of Rsubread package version 2.14.2 (Liao et al., 2014) (http://subread.sourceforge.net) was used to quantitate the number of reads mapped to each gene.
Assembly: GCA_000022165.1
Supplementary files format and content: A tab-delimited text file includes general statistics on the library depth and read alignment.
Supplementary files format and content: A tab-delimited text file include DESeq normalized trnascript coverage values for each sample.
Library strategy: Cappable-Seq
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| Submission date |
Jul 02, 2024 |
| Last update date |
Oct 10, 2024 |
| Contact name |
Alexander S Balkin |
| E-mail(s) |
freelivingresearcher@gmail.com
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| Phone |
0079096071417
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| Organization name |
Institute for Cellular and Intracellular Symbiosis
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| Lab |
Laboratory og Biomedical technologies
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| Street address |
11 Pionerskaya st.
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| City |
Orenburg |
| ZIP/Postal code |
460000 |
| Country |
Russia |
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| Platform ID |
GPL34612 |
| Series (1) |
| GSE271311 |
Transcriptome Cappable-Seq Sequencing Data Set of Gene Expression in Salmonella enterica serovar Typhimurium 14028S inside Acanthamoeba castellanii, and under Oxidative and Starvation Stress Conditions |
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| Relations |
| BioSample |
SAMN42234062 |
| SRA |
SRX25184838 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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