Similar protocols

Pipeline publication

[…] bad) to obtain RNA molecules, from 3 independent series of experiments, each one comprising 9 animals. Control cells obtained as described before, were also used to perform RNA extraction. RNA integrity and concentration were assessed by electrophoresis on a 1% agarose gel and by Qubit analyses. The mRNAs from each experimental replicate (9 animals per replicate) and from controls (3 independent culture growth) were purified from total RNA samples, processed and sequenced using Illumina HiSeq 2500 Platform (Tufts Medical School, Boston )., Approximately 40 million of reads of 100 bp paired-end sequences were obtained for each sample. The resulting FastQ files were analyzed for quality using FastQC (http://www.bioinformatics.babraham.ac.uk./projects/fastqc/). Reads were aligned to the mouse genome reference (GRCm38) to remove any possible host contamination, using Bowtie2 2.2.4 (https://doi.org/10.1038/nmeth.1923). After filtered, the reads were aligned to the reference Paracoccidioides genome, Pb18, obtained in NCBI under ABKI00000000 identifier. The alignment was done by Bowtie2 program, and each gene was quantified by counting of reads by feature using the function multicov from bedtools package. Candidates for differentially expressed genes were identified by statistical package DEGSeq, from the Bioconductor repository, using Fisher's test implemented in the package. Gene features presenting 2.0-fold change cut-off, and p-value < 0.001 were considered regulated and classified., The biologic processes were obtained using the Pedant on MIPS (http://pedant.helmholtz-muenchen.de) which provides a tool to browse and search the Functional Categories (FunCat) of proteins. The heat maps were generated using Multiexperimental Viewer 4.8 (www.tm4.org/mev)., P. brasiliensis yeast cells from infected mouse lung or control cells were collected by centrifugation at 10,000 x g during 5 min and washed by addition of 100 μL of RapiGEST (0.2%v/v) (Waters Corp), followed by 3 washes using ultrapure water, and by the addition of extraction buffer (20 mM Tris-HCl pH 8.8; 2 mM CaCl2). This suspension was distributed in tubes containing glass beads in equal volume of the cell pellet and the suspension was processed on ice in bead beater equipment (BioSpec) during 5 cycles of 30 sec. The cell lysate was centrifuged at 10,000 x g during 15 min at 4°C and the supernatant was quantified for protein content, using the Bradford reagent (Sigma Aldri […]

Pipeline specifications

Software tools FastQC, Bowtie2, BEDTools, DEGseq, TM4
Databases FunCat
Organisms Paracoccidioides lutzii, Mus musculus, Homo sapiens, Paracoccidioides brasiliensis, Saccharomyces cerevisiae
Diseases Infection, Paracoccidioidomycosis