Computational protocol: An Optimized Method for Extracting Bacterial RNA from Mouse Skin Tissue Colonized by Mycobacterium ulcerans

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Protocol publication

[…] (i) Ribosomal RNA (rRNA) removal: rRNA was depleted from the mycobacterial total RNA preparations with the RiboZero Epidemiology Illumina kit, which removes eukaryote and prokaryote rRNA in a single step.(ii) Preparation of RNA-seq libraries: The RNA-seq libraries were prepared with the TruSeq Stranded Total RNA LT Sample Prep kit (Illumina). The quality of all libraries was checked with the DNA-1000 kit (Agilent) on a 2100 Bioanalyzer and quantification was performed with Quant-It assays on a Qubit 1.0 fluorometer (Invitrogen).(iii) RNA-seq: Clusters were generated for the resulting libraries, with Illumina HiSeq SR Cluster Kit v4 reagents. Sequencing was performed with the Illumina HiSeq 2500 system and HiSeq SBS kit v4 reagents. Runs were carried out over 65 cycles, including seven indexing cycles, to obtain 65-bp single-end reads. Sequencing data were processed with Illumina Pipeline software (Casava version 1.9). All 65-bp reads were aligned against the complete genome and plasmid sequences of M. ulcerans (Agy99 strain) obtained from the Burulist database with Bowtie software, and against the mouse genome (GRCm38) obtained from the Ensembl database with STAR software. Data were normalized and analyzed in R, with the Bioconductor packages. […]

Pipeline specifications

Software tools BaseSpace, Bowtie
Databases BuruList
Application RNA-seq analysis
Organisms Mus musculus, Mycobacterium ulcerans, Homo sapiens
Diseases Skin Ulcer, Buruli Ulcer