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Sample GSM2741946

Query DataSets for GSM2741946

Status

Public on Aug 15, 2017

Title

SA_E2

Sample type

SRA

Source name

bacterial cell_sulfanilic acid_exponential phase

Organism

Novosphingobium resinovorum

Characteristics

strain: SA1
carbon source: sulfanilic acid
nitrogen source: sulfanilic acid
growth phase: exponential phase

Growth protocol

N. resinovorum SA1 strain was grown in 250 mL Erlenmeyer flasks in 100 mL minimal salt media (MM) [1], containing 10 mM substrates (either sulfanilic acid (SA), or glucose (Glc)), 25 µg/ml (Sm) and 25 mM MOPS (pH=7.0). Addition of MOPS could stabilize the pH during the cell growth, the pH change/drop was less than 0.2 unit in any cultivation. When glucose was used as carbon source, the medium was supplemented with 10 mM NH4Cl, in the case of SA, the substrate was the sole nitrogen source. The cultures were shaken at 28 °C, 180 rpm and the cell growth was monitored by measuring the OD600nm values. Samples for RNA extraction were taken from exponential and late stationary phase. 1. Perei K, Rákhely G, Kiss I, Polyák B, Kovács KL. Biodegradation of sulfanilic acid by Pseudomonas paucimobilis. Appl Microbiol Biotechnol. 2001;55(1):101-107. http://www.ncbi.nlm.nih.gov/pubmed/11234949.

Extracted molecule

total RNA

Extraction protocol

6 ml samples were taken from exponential and late stationary phase cultures grown on Glc or SA in MM. In each case, two independent biological replicas were used. The samples were harvested (17,226 rcf, 5 min, 4 °C) and immediately frozen in liquid nitrogen and stored at -80°C until RNA isolation. The frozen samples were resuspended in 350 µl Zymo DNA/RNA Shield. The resuspended cells were mixed with 700 µl RLT buffer (Qiagen) (with β-merkaptoetanol) and were transferred into a new tube containing 0.8 g, 0.5 mm Glass Beads (Scientific Industries, SI-BG05). The cells were vortexed for 5 minutes on maximum speed with a Vortex-Genie 2 (Scientific Industries, SI-0236) supplemented with a TurboMix Attachment (Scientific Industries, SI-0564). Following lysis, the mixtures were centrifuged at 9,600 rcf for 10 seconds to remove the glass beads and cell debris. The supernatants were used for purification of total RNAs with the Qiagen RNeasy Mini Kit protocol (Qiagen) with on-column DNase digestion. Then the Purified RNAs were treated again with RNase free DNase I (Sigma) to remove any residual genomic DNA contamination. Finally, the RNAs obtained were repurified with the RNeasy Mini Kit Clean Up protocol (Qiagen). The integrity of the RNA was checked by Agilent 2100 Bioanalyzer.
RNA libraries were prepared for sequencing using standard Illumina epicenter ScriptSeq (Bacteria) protocol

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina MiSeq

Description

SA, exponential phase, rep 2

Data processing

The reads were trimmed using CLC Genomics Workbench version 7 with the following configurations: -- Ambiguous trim = Yes -- Ambiguous limit = 2 -- Quality trim = Yes -- Quality limit = 0.05 -- Create report = Yes -- Save discarded sequences = No -- Remove 5' terminal nucleotides = No -- Discard short reads = No -- Remove 3' terminal nucleotides = No -- Discard long reads = No -- Save broken pairs = No
The trimmed reads were aligned to the CP017075.1, CP017076.1, CP017077.1, CP017078.1, CP017079.1 genome assembly using CLC Genomics Workbench version 7 with the following configurations: -- Reference type = Genome annotated with genes only -- Reference sequence = CP017075-9 -- Gene track = CP017075-9 (CDS) -- Mapping type = Also map to inter-genic regions -- Mismatch cost = 2 -- Insertion cost = 3 -- Deletion cost = 3 -- Length fraction = 0.8 -- Similarity fraction = 0.8 -- Global alignment = No -- Auto-detect paired distances = Yes -- Strand specific = Both -- Maximum number of hits for a read = 20 -- Count paired reads as two = No -- Expression value = Total counts -- Calculate RPKM for genes without transcripts = No -- Create report = Yes -- Create fusion gene table = No -- Create list of unmapped reads = No
Differential gene expression analysis using EdgeR
Genome_build: CP017075.1, CP017076.1, CP017077.1, CP017078.1, CP017079.1
Supplementary_files_format_and_content: Excel file "Full_Datasets_DEG_Annotations.xlsx" contains the annotation dates, normalized expression values, log2 fold change values, False Discovery Rate adjusted p-values and RPKM values of the genes in the 3 examined conditions: Glc_S vs. Glc_E, SA_E vs. Glc_E, SA_S vs. SA_E.

Submission date

Aug 14, 2017

Last update date

May 15, 2019

Contact name

Botond Hegedüs

E-mail(s)

h.botond@outlook.com

Organization name

University of Szeged

Department

Department of Biotechnology