Computational protocol: Interaction of Salmonella enterica Serovar Typhimurium with Intestinal Organoids Derived from Human Induced Pluripotent Stem Cells

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Protocol publication

[…] RNA was prepared from iHOs microinjected with S. Typhimurium SL1344 and iHOs microinjected with PBS (as described above) for three biological replicates per condition. Multiplexed mRNA libraries were prepared by using the Illumina TruSeq protocol and sequenced via paired-end sequencing with the Illumina-C HiSeq 2500 platform. Each lane of Illumina sequence was assessed for quality on the basis of GC content, average base quality, and adapter contamination. RNA-Seq reads were aligned with TopHat () version 2.0.8 with the human reference version GRCh37 used for the 1000 Genomes project. The read counts per gene were generated with featureCounts version 1.4.5-p1. The annotation for featureCounts came from ENSEMBL 75. Read counts were used to represent gene expression levels. R version 3.1.2 was used to import count data, and the DESeq2 package was used to normalize the data and detect differentially expressed genes (). Significantly differentially expressed genes were selected with a cutoff false-discovery rate of less than 0.05 and a log change of >2.0-fold. Heat maps and principal-component analysis (PCA) plots were constructed from rlog-transformed data with the ggplot2 R library (http://cran.r-project.org/web/packages/ggplot2/index.html). For the 250 most significantly upregulated genes, enriched gene ontology terms were identified by using InnateDB with the category Biological Processes selected. […]

Pipeline specifications

Software tools TopHat, Subread, DESeq2, Ggplot2
Databases InnateDB
Applications Miscellaneous, RNA-seq analysis
Organisms Salmonella enterica subsp. enterica serovar Typhimurium, Homo sapiens, Escherichia coli, Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344
Diseases Infection