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Sample GSM4318424

Query DataSets for GSM4318424

Status

Public on May 01, 2020

Title

∆NtrYX Rep A

Sample type

SRA

Source name

Bacterial culture

Organism

Cereibacter sphaeroides 2.4.1

Characteristics

media: SIS medum
oxygen: 30% Oxygen
tissue: Bacterial culture

Growth protocol

R. sphaeroides 2.4.1 cultures were grown in Sistrom’s minimal medium (SMM), with 4 g/L succinate. 500 ml cultures were grown in Roux bottles bubbled with 30% O2, 1% CO2, and 69% N2.

Extracted molecule

total RNA

Extraction protocol

R. sphaeroides cultures were grown aerobically in 500 ml cultures. At OD600 = 0.3-0.4, 44 ml of harvested culture was combined with 6 ml of ice-cold stop solution (95% ethanol, 5% water-saturated phenol). These mixtures were centrifuged at 4 °C for 10 min at 5,000 x g. Cell pellets were resuspended into 2 ml of lysis solution (2% SDS, 16 mM EDTA in RNase-free water) and then incubated at 65 °C for 5 min. Nucleic acid was extracted by adding equal volume of acid-phenol:chloroform (5:1, pH4.5), mixing, incubating at 65 °C for 5 min, mixing, and centrifuging at 16,000 x g. The aqueous phase was removed, and extracted 2 more times, before adding an equal volume chloroform, and centrifuging at 16,000 x g for 5 min. 1/10 volume of 3M sodium acetate and 1 ml isopropanol was added to the aqueous phase, followed by incubation at -20°C for 30 min, and then centrifugation at 16,000 x g for 30 min at 4°C to precipitate the nucleic acids. The pellet was washed with 75% ethanol and resuspended in RNase-free water. Samples were treated with RNase-free DNase and then purified with an RNeasy kit (Qiagen).
RNA-seq library preparation and sequencing was performed at the Joint Genome Institute. Libraries for sequencing were created using the Illumina TruSeq Stranded Total RNA kit (Illumina) following the standard protocol. RNA-seq libraries were sequenced on an Illumina NextSeq in 2x151 reads using the standard protocol.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

For RNA-seq analysis, the paired-end FASTQ files were split into R1 and R2 files and R1 files were retained for further analysis as previous data contained only single-end reads. 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 R. sphaeroides 2.4.1 genome sequence (GenBank accession GCA_000012905.2) using Bowtie2 version 2.2.2 with default settings except the number of mismatches was set to 1. Aligned reads were mapped to gene locations using HTSeq version 0.6.0 using default settings except for the “reverse” strandedness argument was used. edgeR version 3.26.8 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. Raw sequencing reads were normalized using the reads per kilobase per million mapped reads (RPKM).
Genome_build: GCA_000012905.2
Supplementary_files_format_and_content: Normalized RPKM read counts for RNA-seq. WIG files for ChIP-seq

Submission date

Feb 18, 2020

Last update date

May 01, 2020

Contact name

Kevin S Myers

E-mail(s)

kmyers2@wisc.edu

Organization name

University of Wisconsin - Madison

Department

Great Lakes Bioenergy Research Center