DOI: 10.5061/dryad.1jwstqk2g
title: CompareRhythms data in healthy and asthmatic airway epithelial cells

description: Rationale: Cellular circadian rhythms regulate multiple cellular processes
 and have been hypothesized to influence epithelial function in asthma.
 Airway resistance has circadian variability in patients with asthma,
 profiling of circadian rhythms of gene expression in human airway
 epithelia has not been performed. Organotypic cultures of primary human
 airway epithelial cells differentiated at an air-liquid interface
 recapitulate the major cell types of human airway epithelia to allow for
 evaluation of gene expression not possible in vivo and demonstrate
 rhythmicity of core circadian genes after temperature synchronization.
 Methods:  Primary human airway epithelial cells from 6 healthy
 children and 6 children with asthma were differentiated an air-liquid
 interface and circadian rhythms were synchronized using cycled incubator
 temperature. RNA was harvested every 4 hours and RNA-sequencing performed
 to measure transcriptome-wide expression, with differential rhythmicity of
 genes identified using CompareRhythms. Results: Core circadian genes ARNTL
 and NR1D1 demonstrated rhythmicity in both healthy and asthmatic airway
 epithelial cells with maintained phase relationships. In RNAseq data from
 healthy and asthmatic airway epithelial cells, 646 (4%) of protein-coding
 genes were rhythmic, with 110 genes exhibiting differential rhythmicity in
 asthma. Gene set enrichment analysis using EnrichR revealed that genes in
 circadian rhythm, nuclear receptor, and cell adhesion pathways were
 rhythmic. Neutrophil chemotaxis, cytokine mediated signaling, and viral
 protein interactions with cytokine receptor pathways demonstrated
 differential (gain, loss, or change) rhythmicity in asthma. Conclusions:
 Core circadian rhythm genes maintain rhythmicity in healthy and asthmatic
 human airway epithelia, and regulate cytokine mediated signaling and
 neutrophil chemotaxis pathways in asthmatic human airway epithelia.
description: AECs from healthy children and children with physician-diagnosed
 allergic asthma were obtained from subjects (ages 6-16) while under
 general anesthesia for elective procedures. AECs were expanded and at
 passage 3 were differentiated at an ALI in PneumaCult ALI media (Stemcell)
 at 37°C, producing an organotypic differentiated epithelial culture with
 mucociliary morphology. AECs from children were obtained under study
 #12490 and #1596 approved by the Seattle Children’s Hospital Institutional
 Review Board with investigations conducted following the rules of the
 Declaration of Helsinki of 1975. Temperature cycled synchronization
 occurred for 6 days (12 hours at 37°C, 12 hours at 34°C), then cultures
 were maintained at a constant 37°C for 48 hours during RNA isolation. RNA
 was collected every 4 hours for 48 hours, beginning 4 hours after final
 media change and completion of temperature synchronization. Two
 independent cultures were harvested for RNA at each timepoint. Samples
 with RIN &gt;8 were prepared using poly-A library preparation by
 Novogene sequencing using 150 base paired-end reads with targeting 20-30
 million reads per sample performed using Illumina sequencing. STAR
 (version 2.4.2a) was used to align reads after adapter trimming to the
 Ensembl version of the human genome (GRCh38, Ensembl 91). For quality
 control, samples that had human aligned counts greater than 5 million
 mapped reads and a median coefficient of variation coverage less than 0.9
 were kept. No samples failed quality control. Genes were filtered to
 include those that had at least 0.1 counts per million in at least 40
 samples and further filtered for protein coding genes. Counts were then
 normalized using the trimmed mean of M (TMM) approach in the edgeR
 package. In R, the package CompareRhythms using the cosinor function was
 used to characterize rhythmicity with FDR adjusted p-value of 0.05,
 minimum amplitude 0.2, period length of 24, and an FDR cutoff of 0.2 for
 differential rhythmicity.
description: # CompareRhythms Data in healthy and asthmatic airway epithelial cells
 [https://doi.org/10.5061/dryad.1jwstqk2g](https://doi.org/10.5061/dryad.1jwstqk2g) This dataset contains the CompareRhythms input and output data on transcriptomic analyses performed in cultured primary human airway epithelial cells following temperature synchronization. ## Description of the data and file structure AECs from healthy children and children with physician-diagnosed allergic asthma were obtained from subjects (ages 6-16) while under general anesthesia for elective procedures. AECs were expanded and at passage 3 were differentiated at an ALI in PneumaCult ALI media (Stemcell) at 37°C, producing an organotypic differentiated epithelial culture with mucociliary morphology. AECs from children were obtained under study #12490 and #1596 approved by the Seattle Children’s Hospital Institutional Review Board with investigations conducted following the rules of the Declaration of Helsinki of 1975. Temperature cycled synchronization occurred for 6 days (12 hours at 37°C, 12 hours at 34°C), then cultures were maintained at a constant 37°C for 48 hours during RNA isolation. RNA was collected every 4 hours for 48 hours, beginning 4 hours after final media change and completion of temperature synchronization. Two independent cultures were harvested for RNA at each timepoint. Samples with RIN &gt;8 were prepared using poly-A library preparation by Novogene sequencing using 150 base paired-end reads with targeting 20-30 million reads per sample performed using Illumina sequencing. STAR (version 2.4.2a) was used to align reads after adapter trimming to the Ensembl version of the human genome (GRCh38, Ensembl 91). For quality control, samples that had human aligned counts greater than 5 million mapped reads and a median coefficient of variation coverage less than 0.9 were kept. No samples failed quality control. Genes were filtered to include those that had at least 0.1 counts per million in at least 40 samples and further filtered for protein coding genes. Counts were then normalized using the trimmed mean of M (TMM) approach in the edgeR package. In R, the package CompareRhythms using the cosinor function was used to characterize rhythmicity with FDR adjusted p-value of 0.05, minimum amplitude 0.2, period length of 24, and an FDR cutoff of 0.2 for differential rhythmicity. The input file is a matrix ensembl gene ID in the first column, and individual samples with sample ID_time_replicate as names. The normalized counts were generated as above. The CompareRhythms package [https://doi.org/10.5281/zenodo.7699722](https://doi.org/10.5281/zenodo.7699722) outputs a matrix with the following: id = Ensembl Gene ID category = CompareRhythms categorization of "same" "loss" "change" or "gain" in asthma as compared to healthy rhythmic_in_ctrl = True/False for rhythmic expression in healthy rhythmic_in_asthma = True/False for rhythmic expression in asthma diff_rhythmic = True/False for rhythmic change in asthma as compared to healthy ctrl_amp = amplitude output from cosinor analysis in healthy ctrl_phase = phase calculation from cosinor analysis in healthy asthma_amp = amplitude output from cosinor analysis in asthma asthma_phase = phase calculation from cosinor analysis in asthma p_val_ctrl_or_b = p-value for rhythmicity in healthy or asthma adj_p_val_ctrl_or_assthma = adjusted p-value for multiple comparison testing p_val_DR = p-value for differential rhythmicity comparing healthy vs asthma adj_p_val_DR = adjusted p-value for differential rhythmicity for multiple comparison testing