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Sample GSM4289013

Query DataSets for GSM4289013

Status

Public on Jun 01, 2020

Title

22 µM DO_1

Sample type

SRA

Source name

cells

Organism

Candidatus Thioglobus autotrophicus

Characteristics

strain: EF1
dissolved oxygen (do) um: 22

Treatment protocol

One set of triplicate bottles was kept anoxic. A second set of bottles received 1 mL of filtered-sterilized air (approximate initial aqueous DO concentration = 3.8 μM), and a third set received 5 mL of filter-sterilized air (approximate initial aqueous DO concentration = 22 μM). All bottles were incubated without shaking at 13ºC during the experiments.

Growth protocol

Filter-sterilized Puget Sound seawater (PS media; collected June 2017) was used as bacterial growth media with filter-sterilized thiosulfate (1 mM) added as the energy source. The PS media initially contained 13.5 ± 0.58 μM nitrate. No additional nitrate was added to the media. PS media (100 mL) was placed into acid-washed (10% HCl) and autoclaved 160 mL glass serum bottles and sealed with 20 mm blue butyl rubber stoppers and aluminum crimp caps (DWK Life Sciences, Millville, NJ). Sealed serum bottles were subsequently purged (7 minutes liquid, followed by 5 min headspace) with a mixture of nitrogen gas containing 1000 ppm carbon dioxide to remove dissolve oxygen (DO). Purged anoxic PS media bottles were prepared in triplicate.

Extracted molecule

total RNA

Extraction protocol

When EF1 cell density reached approximately 1-3x106 cells/ml, bottles were sacrificed for RNA extraction. Cultures (~100 mL) were vacuum filtered onto 47 mm 0.2 μm IsoporeTM membrane filters (Millipore Inc., Billerica, MA, USA) under a nitrogen headspace to limit oxygen diffusion. Filters were placed into a 50 ml conical tube containing Trizol reagent (3 mL), 375 μL glass and zirconia bead mixture, and 12.5 μl (6.79×109 copies) internal mRNA standards. Bead-beating was performed for 5 minutes on a vortexer. After a 1 min centrifugation step at 3,220 xg, RNA was extracted from the supernatant with the Zymo Direct-zol RNA MiniPrep Plus kit (Zymo Research Corporation, Irvine, CA) according to the manufacturer’s protocol (including on-column DNase I treatment). To further minimize DNA contamination, RNA extracts were subjected to a separate DNase I treatment step with the TURBO DNA-freeTM kit (Thermofisher Scientific, Waltham, MA). RNA concentrations were estimated with the QubitTM fluorometer and HS RNA assay kit (Thermofisher Scientific,Waltham, MA)
rRNA was depleted from the extractions with the Ribozero rRNA removal kit for bacteria (Illumina, Inc.San Diego, CA). Libraries were prepared from the depleted RNA using the Illumina Truseq library prep kit v2, normalized and loaded at equal concentration on the Illumina MiSeq using V2 chemistry and then subjected to PE150 sequencing at the University of Washington

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina MiSeq

Data processing

Raw sequencing data was processed online with software available on the Galaxy server (usegalaxy.org). First, fastq data files were converted to fastqsanger format with the fastq groomer tool. The quality of transcriptomic sequence reads was assessed with FastQC.
Forward strand transcriptomic reads were then mapped to the T. autotrophicus strain EF1 genome sequence (Genbank accession no. CP010552) using HISAT2. This resulted in 6.97×105– 1.38×106 reads mapped to the EF1genome per treatment. Similarly, forward strand reads were also mapped to the internal mRNA standards, yielding 1.41×105 – 2.48×105 mRNA standard reads per treatment (9-22% of total reads).
HISAT2-generated BAM files for each treatment were loaded into ReadXplorer 2.2.3 where differential gene expression analyses were performed. First, a transcript count table was generated. The percent recovery of internal mRNA standards was used to estimate the absolute number of EF1 transcripts per treatment and subsequently normalized to the number of EF1 cells per RNA extraction to obtain absolute “transcript per cell” values
Differential gene expression analysis among the three different DO treatments was performed with the DeSeq2 algorithm. Genes that were differentially expressed at a statistically significant level (p<0.05) were considered for further analysis
Genome_build: T. autotrophicus strain EF1 genome sequence (Genbank accession no. CP010552)
Supplementary_files_format_and_content: excel files inlcude transcript counts, cell counts and transcript per cell values for each gene in the genome. DeSeq2 output provided in excel files.

Submission date

Jan 29, 2020

Last update date

Jun 01, 2020

Contact name

Timothy E Mattes

E-mail(s)

tim-mattes@uiowa.edu

Phone

319-335-5065

Organization name

University of Iowa