Sequence Libraries Preparation from Total RNA

Total RNA was extracted with the TRI reagent (Ambion) using the manufacturer’s instructions. The RNase inhibitor RiboLock (Fermentas) was added to the samples and DNA was removed by treatment with DNase I (Fermentas). The integrity of the RNA samples was assessed with an Agilent 2100 Bioanalyzer (Agilent Technologies). Subsequently, the 23S, 16S, and 5S rRNAs were removed by subtractive hybridization using the MICROBExpress kit (Ambion) following the protocol reported by Gómez-Lozano et al. (2014) (link). Removal of the rRNA was confirmed by an analysis with an Agilent 2100 Bioanalyzer (Agilent Technologies). Sequencing libraries were prepared using the TruSeq Stranded mRNA Sample Preparation kit (Illumina). After each step, the samples were validated using an Agilent 2100 Bioanalyzer (Agilent Technologies) and the final RNA concentration was measured using a Qubit 2.0 Fluorometer (Invitrogen). The libraries were sequenced using the Illumina HiSeq2000 platform with a paired-end protocol and read lengths of 100 nucleotides.

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Rubio-Gómez J.M., Santiago C.M., Udaondo Z., Garitaonaindia M.T., Krell T., Ramos J.L, & Daddaoua A. (2020). Full Transcriptomic Response of Pseudomonas aeruginosa to an Inulin-Derived Fructooligosaccharide. Frontiers in Microbiology, 11, 202.

Publication 2020

TRI reagent RiboLock DNase I Agilent 2100 Bioanalyzer MICROBExpress kit TruSeq Stranded mRNA Sample Preparation kit Qubit 2.0 Fluorometer HiSeq2000 platform

5s rrnas Dnase i Mrna Nucleotides Rnase Rrna Subtractive hybridization

Corresponding Organization :

Other organizations : Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Universidad de Granada, Universidad de Málaga, Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora", University of Arkansas for Medical Sciences, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas

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Variable analysis

independent variables

Treatment with DNase I
Subtractive hybridization using the MICROBExpress kit

dependent variables

Integrity of the RNA samples
Removal of the 23S, 16S, and 5S rRNAs
Final RNA concentration

control variables

Use of the TRI reagent (Ambion) for RNA extraction
Addition of the RNase inhibitor RiboLock (Fermentas)
Use of the Agilent 2100 Bioanalyzer (Agilent Technologies) for RNA analysis
Use of the Qubit 2.0 Fluorometer (Invitrogen) for RNA quantification
Use of the TruSeq Stranded mRNA Sample Preparation kit (Illumina) for library preparation
Sequencing on the Illumina HiSeq2000 platform with a paired-end protocol and read lengths of 100 nucleotides

controls

Positive control: Not specified
Negative control: Not specified

Annotations

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