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Sample GSM8114843

Query DataSets for GSM8114843

Status

Public on Feb 29, 2024

Title

Vanillin Rep 1

Sample type

SRA

Source name

Δ1879 (sacB) Δ2819 (ligI) Δ2864 (desC) Δ2865 (desD)

Organism

Novosphingobium aromaticivorans DSM 12444

Characteristics

strain: DSM 12444
cell type: bacterial
treatment: Vanillin

Treatment protocol

Growth was stopped by the 1:8 addition of ice cold 5% acid phenol:chloroform (5:1) in ethanol

Growth protocol

Four isolated N. aromaticivorans PDC12444 colonies were cultured and grown overnight. The next day, the overnight cultures were diluted 1:1 with SMB minimal medium supplemented with 1g/L glucose and grown for one hour. The cultures were then diluted 1:100 into separate cultures of SMB minimal medium supplemented with 1g/L glucose, 1g/L glucose plus 0.5mM DC-A, 1g/L glucose plus 0.5mM vanillin, or 1g/L glucose plus 0.5mM ferulic acid. These cultures were grown until they reached mid-exponential growth phase.

Extracted molecule

total RNA

Extraction protocol

The cells were pelleted by centrifugation (4,300 x g for 10 minutes) at 4°C and stored at -80°C. Cell pellets were resuspended in 2 mL of lysis solution (2% sodium dodecyl sulfate, 16mM EDTA, RNase-free water) and heated at 65°C for 5 minutes. Nucleic acids were extracted using 2 mL of acidic phenol:chloroform heated to 65°C, mixed by inversion, incubated at 65°C for 5 minutes, and centrifuged at room temperature for 7 minutes at 20,000 x g. The aqueous phase was removed, extracted twice more, before addition of 2 mL of chloroform, mixed, and centrifuged once more at room temperature for 7 minutes at 20,000 x g. The aqueous layer was removed and mixed with 1/10 volume of 3M sodium acetate and equal volume of isopropanol, and incubated at -20°C for >1 hour to precipitate nucleic acids that were collected by centrifugation at 4°C for 30 minutes at 16,000 x g. The pellet was washed twice with 1 mL of 75% EtOH (prepared with RNase-free water). Once residual ethanol was removed by evaporation, the pellet was resuspended in 85 μL of RNase-free water. Samples were treated with RNase-free DNase (10 μL 10x DNase buffer, 2 μL DNase, 3 μL RNasin (Promega)) and purified using RNeasy kit (Qiagen).
RNA-seq library preparation and sequencing was performed by the Joint Genome Institute (JGI) using default parameters. rRNA in the samples was depleted using the QIAseq FastSelect kit (Qiagen, Germantown, MD). Libraries were constructed using the TruSeq stranded mRNA kit (Illumina) following standard JGI protocols.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

Vanillin

Data processing

All paired-end FASTQ files were processed through the same pipeline. Reads were trimmed using Trimmomatic version 0.3 with the default settings except for a HEADCROP of 5, LEADING of 3, TRAILING of 3, SLIDINGWINDOW of 3:30, and MINLEN of 36. After trimming, the reads were aligned to the N. aromaticivorans DSM12444 genome sequence (GenBank accession GCF_000013325.1) using bwa-mem (version 0.7.17-h5bf99c6_8) with default settings. Alignment files were further processed with Picard-tools (version 2.26.10) (https://broadinstitute.github.io/picard/) (CleanSAM and AddOrRepleaceReadGroups commands) and samtools (version 1.2) (sort and index commands). Paired aligned reads were mapped to gene locations using HTSeq version 0.6.0. The R package edgeR (version 3.30.3) with default settings was used to identify significantly differentially expressed genes from pairwise analyses, using Benjamini and Hochberg false discovery rate (FDR) less than 0.05 as a significance threshold (57). Raw sequencing reads were normalized using the fragments per kilobase per million mapped reads method (FPKM).
Assembly: GCF_000013325.1
Supplementary files format and content: Metz_htseq_counts.txt contains the RAW gene counts for the experiments as determined by Htseq-count
Supplementary files format and content: Metz_FPKM_results.xlsx contains the FPKM normalized read counts for all experiments

Submission date

Feb 27, 2024

Last update date

Feb 29, 2024

Contact name

Kevin S Myers

E-mail(s)

kmyers2@wisc.edu

Organization name

University of Wisconsin - Madison

Department

Great Lakes Bioenergy Research Center