Computational protocol: Characterization of the Probiotic Yeast Saccharomyces boulardii in the Healthy Mucosal Immune System

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Protocol publication

[…] RNA extraction of MLNs from naïve and S. boulardii-treated C57BL/6J mice was performed using the Qiagen RNeasy mini kit with DNase treatment according to manufacturer’s protocols. Sample quality analyses, library preparation, and sequencing were performed by the Huntsman Cancer Institute High Throughput Genomics Core (University of Utah). RNA integrity was confirmed using an Agilent RNA ScreenTape assay, and only high quality RNA (RIN >8.0) was submitted for further processing. Library preparation with oligo dT selection was performed using the Illumina TruSeq Stranded mRNA Sample Preparation Kit. Sequencing libraries (25 pM) were chemically denatured and applied to an Illumina HiSeq v4 single read flow cell using an Illumina cBot. Single end sequencing of 50 bp reads was performed using an Illumina HiSeq 2000 according to standard protocols.A mean of 41.1 million reads per sample were acquired, with very high quality as assessed by FastQC (Babraham Institute) (). Reads were mapped to the GRCm38 Mus musculus genome (accessed via Ensembl []) using TopHat2 []. HT-seq [] count was used to assign aligned reads to genes from the Ensembl release 82 GRCm38 genome annotation. Differential expression analysis, MA plots, and clustering were performed with DESeq2 []. Genes with a p-value (adjusted for multiple corrections) of 0.05 or less were considered differentially expressed. Principal component analysis was performed with two components on the log-transformed expression of the 1,000 genes with highest variance among samples using the R package psych. RNA-seq reads have been deposited to the NCBI Sequence Read Archive (SRA) under accession number SRP067985. […]

Pipeline specifications

Software tools FastQC, TopHat, HTSeq, DESeq2
Application RNA-seq analysis
Organisms Saccharomyces cerevisiae, Mus musculus
Diseases Communicable Diseases