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Sample GSM3502728

Query DataSets for GSM3502728

Status

Public on Jul 03, 2019

Title

T3 Replicate 2

Sample type

SRA

Source name

bacterial culture

Organism

Nitrospira moscoviensis

Characteristics

time point: T3, day 127

Growth protocol

Cells were grown in NOB mineral salts medium for lithoautotrophic growth as described in Spieck and Lipski 2010 with the following modifications: CaCO3 was replaced with CaCl2·2 H2O in the same concentration and the following trace element composition was used per liter of medium, 34.4 µg of MnSO4·1 H2O, 50 µg of H3BO3, 70 µg of ZnCl2, 72.6 µg of Na2MoO4·2 H2O, 20 µg of CuCl2·2 H2O, 24 µg of NiCl2·6 H2O, 80 µg of CoCl2·6 H2O, and 2000 µg of FeSO4·7 H2O.N. moscoviensis was cultivated in a 7 L glass bioreactor inoculated with an active batch culture. The bioreactor was operated at a 5 L working volume and was equipped with pH, dissolved oxygen, temperature and a level sensors, all connected to a biocontroller (bioreactor and equipment all by Applikon Biotechnology B.V., Schiedam, the Netherlands). The controller setup automatically maintained pH at 7.7 by constant flushing with Ar/CO2 (95%/5% v/v) and buffering with a 1 M KHCO3 solution. Dissolved oxygen was kept at 30% by providing filtered air or N2 gas through an aeration tube with sparger. The temperature was upheld at 39 °C by using a loop type heat exchanger. CO2 levels were controlled manually. Ar/CO2 (95%/5% v/v) flow was increased whenever CO2 was no longer in excess and acidifying the medium as indicated by the use of 1 M KHCO3 solution. The medium was constantly stirred at 150 rpm with a marine style impeller. All medium and solutions were sterilized by autoclavation or sterile filtration prior to use and the reactor operated aseptically to retain the purity of the culture.

Extracted molecule

total RNA

Extraction protocol

Transcriptome analyses were performed using three technical replicates for each of the three sampling points. Biomass of 70 ml culture volume was harvested by centrifugation at 20,000 g, 4 °C, 10-20 min. RNA was immediately extracted using TRIzol reagent (Thermo Fisher Scientific) following the manufacturer’s protocol with the following modifications: all incubation and centrifugation times were doubled, RNA was precipitation overnight at –20 °C and afterwards centrifuge at 12,000 g, 4 °C, 20-30 min to collect RNA, no heating step for RNA resuspension was performed. Residual genomic DNA was removed with a DNase I (Thermo Fisher Scientific) treatment following the manufacturer’s protocol. Purity of RNA was confirmed on an ethidium bromide stained 0.9% agarose gel. The MEGAclear kit (Thermo Fisher Scientific) was used following the manufacturer’s protocol to purify the RNA and remove small RNAs. RNA quantity was controlled using the Qubit RNA HS Assay Kit (Thermo Fisher Scientific). Next, ribosomal RNA was depleted using the MICROBExpress kit (Thermo Fisher Scientific) following the manufacturer’s protocol.
Transcriptome libraries were prepared with the Ion Total RNA-Seq Kit v2 (Ion Torrent by Thermo Fisher Scientific). RNA yield and size distribution were analyzed using a 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Ion Torrent PGM

Description

bioreactor culture grown on nitrite
Read_counts.csv
RPKM_DESeq_Wald_statistics.csv

Data processing

Raw reads from IonTorrent PGM sequencing were quality filtered using the CLC genomics workbench (CLC bio, Qiagen, Hilden, Germany) based on a minimum quality score of 0.05, a maximum sequencing length of 300 bp, and allowing for two ambiguous nucleotides.
Filtered reads were mapped to the N. moscoviensis genome (accession number NZ_CP011801.1) using BBMap v35.92 (Bushnell, 2015) and counted using featureCounts (Liao et al 2014) with the parameters ‘minid-0.95’ with ‘fracOverlap-0.9’ for a minimum alignment identity of 95% over 90% of the read length and ‘ambig=random’.
Differential gene expression analysis was performed using the DESeq2 package (Love et al., 2014) in R. Differential expression over multiple timepoints was tested using the likelihood ratio test. Differential expression between timepoints was tested using the Wald test. Gene expression levels were compared by ranking the CDSs based on their reads per kilobase per million reads (RPKM) values and the log2-fold to median was calculated.
Genome_build: genome accession number NZ_CP011801.1

Submission date

Dec 06, 2018

Last update date

Jul 03, 2019

Contact name

Aniela Mundinger

E-mail(s)

a.mundinger@science.ru.nl

Organization name

Radboud University

Street address

Heyendaalseweg 135