Computational protocol: Overexpression of Differentially Expressed Genes Identified in Non-pathogenic and Pathogenic Entamoeba histolytica Clones Allow Identification of New Pathogenicity Factors Involved in Amoebic Liver Abscess Formation

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Protocol publication

[…] RNA for RNA-Seq library preparation was purified as described above. RNA quantity and quality were evaluated spectrophotometrically (NanoDrop 2000, Thermo Fisher Scientific, Schwerte, Germany) and with an Agilent 2100 Bioanalyzer with RNA 6000 Pico Assays kit (Agilent Technologies, Waldbronn, Germany).Samples were Turbo DNase-treated using TURBO DNA-free Kit (Ambion-Thermo Fisher Scientific, Schwerte, Germany). After quality control, rRNA was depleted using RiboZero Magnetic Gold kit (Human/Mouse/Rat; Epicentre-Illumina, Munich, Germany) and Agencourt RNAClean XP kit (Beckmann Coulter, Krefeld, Germany), according to the manufacturers’ protocol. RNA-Seq libraries were then generated using ScriptSeq v2 kit (Epicentre-Illumina, Munich, Germany) according to the manufacturer’s instructions. Each library was indexed with Illumina-compatible barcodes to allow multiplexing. The individual libraries were assessed using Qubit dsDNA high sensitivity kit and Bioanalyzer DNA HS chips to ascertain the concentration (4 nM) and fragment size distribution, respectively, prior to library multiplexing. Libraries were denatured and diluted to final concentration of 8 pM for sequencing on the MiSeq platform following the manufacturer’s instructions. Reads were aligned to E. histolytica transcriptome (AmoebaDB 28, released 30 March 2016) using Bowtie 2 version 2.2.3 [] and differential expression was analysed using DESeq [] version 1.18. […]

Pipeline specifications

Software tools Bowtie, DESeq
Databases AmoebaDB
Application RNA-seq analysis
Organisms Entamoeba histolytica, Rattus norvegicus
Diseases Liver Abscess, Liver Abscess, Amebic
Chemicals Ethanol