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Sample GSM7688818

Query DataSets for GSM7688818

Status

Public on Aug 12, 2023

Title

∆vtlR/lsrB, 24h, biol rep2

Sample type

SRA

Source name

bacteria

Organism

Agrobacterium fabrum

Characteristics

strain: C58
cell type: bacteria
genotype: deltavtlR/lsrB (deltaatu2186)
treatment: Grown for 24h in YEB medium at 30 °C.

Growth protocol

Cells were grown at 30 °C in YEB medium. Samples were taken after 24h of incubation.

Extracted molecule

total RNA

Extraction protocol

RNA was extracted via hot-phenol method as described in Wilms et al. (2012, DOI: 10.4161/rna.17212). Samples were treated with RNase-free DNase.
RNA libraries were prepared by Novogene using 1 µg of RNA and the NEBNext® Ultra™ RNA Library Prep Kit for Illumina®. Index codes were added to attribute sequences to each sample. The RNA was fragmented with divalent cations in NEBNext First Strand Synthesis Reaction Buffer (5x) at elevated temperature. Then, first strand synthesis was carried out with random hexamer primers and M-MuLV reverse transcriptase. Second strand synthesis was performed with DNA polymerase I and RNase H. Blunt ends were created from overhangs via exonuclease/polymerase activities. DNA 3' ends were adenylated and NEBNext Adaptors with hairpin loop were ligated. Fragments were purified with the AMPure XP system. The cDNA was then incubated at 37 °C for 15 minutes with 3 µl USER Enzyme and then at 95 °C for 5 minutes. The subsequent PCR was performed with Phusion High-Fidelity DNA polymerase, Universail PCR primers, and Index (X) Primer. The PCR products were purified via AMPure XP system and the quality was assured via Agilent Bioanalyzer 2100.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

Adapter sequences were removed via cutadapt 1.18 with Python 3.7.9 (options: -j 4 -q 10)
Cleaned reads were mapped with bwa (0.7.17-r1188) mem on default settings.
Duplicate reads were removed with Samtools 1.10.
Differential gene expression was analyzed with DESeq2. A maximal adjusted p value of 0.05 was used as threshold.
Assembly: NCBI ID: 13606; 671 small RNA genes were added from Wilms et al. (2012, DOI: 10.4161/rna.17212), Lee et al. (2013, DOI: 10.1371/journal.pone.0070720) and from Möller et al. (2014, DOI: 10.1371/journal.pone.0110427)
Supplementary files format and content: Files include the binary logarithm of the fold change of each gene in the mutant and the corresponding p values.

Submission date

Aug 07, 2023

Last update date

Aug 12, 2023

Contact name

Franz Narberhaus

E-mail(s)

franz.narberhaus@rub.de

Organization name

Ruhr University Bochum

Department

Microbial Biology