IL-5 induced gene expression in human differentiated airway epithelial (ALI) culture

RNA sequencing was performed on differentiated airway epithelial cells that either remained untreated or were treated with IL-5 for 6 or 24 hours. Total RNA samples were sent to the DNA Sequencing Facility at the University of Wisconsin-Madison Biotechnology Center's Gene Expression Center for library preparation and sequencing. RNA was quantified using NanoDrop and Agilent NanoChip. Library preparation was based on the TruSeq RNA Sample Preparation v2 Guide. cDNA libraries were assayed on Agilent DNA1000 Chip to verify library size. Genes were filtered to include those that had a trimmed mean of M values (TMM) normalization count of at least 1 in at least 10% of libraries and were classified as protein coding using BioMart, and then transformed to log2 counts per million along with observations level weights using voomWithQualityWeights from the limma R package. Normalized voom counts were then used to compare among the 3 conditions by linear modeling (limma) including a random effect for donor. Multiple testing correction was by the Benjamini–Hochberg procedure controlling the false discovery rate (FDR) at level 0.05. Overall design: IL-5 induced gene expression in human differentiated airway epithelial (ALI) culture. Sequencing was performed on differentiated airway epithelial cells that either remained untreated or were treated with IL-5 for 6 or 24 hours.