Three gene lists were created with each containing the closest genes within 2 kb downstream of the ATAC-Seq ROCs which were: 1) differentially accessible between the BRSV challenged and control calves in both Diffbind’s EdgeR and DeSeq2 analyses, 2) uniquely present in the BRSV challenged calves, and 3) uniquely present in the control calves. These three gene lists were separately input into ClusterProfiler (version v3.14.0) [56] in R (version 3.6.1), for DAVID (version 6.8) pathway and GO analysis [5759] using the “EnrichDAVID” function. The annotation types interrogated included: “GOTERM_BP_ALL”, “GOTERM_MF_ALL”, “GOTERM_CC_ALL” and “KEGG_PATHWAY”. Pathways and GO terms from the DAVID ClusterProfiler analyses were considered enriched at a P-value of less than 0.05 and an FDR of 5%.

The three gene lists were merged in R (version 3.6.1) with the genes found to be differentially expressed in the RNA-Seq dataset (P < 0.05, FDR < 0.1, FC > 2) [22], to obtain three new lists comprising only the genes which were differentially expressed in the BLN. These genes, along with their corresponding RNA-Seq fold changes, FDR and P-values were examined for over-represented pathways, cellular and molecular functions, using the Ingenuity Pathway Analysis (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuitypathway-analysis), according to the manufacturer’s instructions [60]. Within IPA, Fisher’s exact test was used with the Benjamini-Hochberg correction for multiple testing for the identification of over-represented pathways and over-represented molecular and cellular functions with a FDR of 10%. Additionally, IPA’s regulation Z-score algorithm, which predicts increases or decreases in functions based on directional changes in the differentially expressed genes was used to predict differences in the over-represented cellular and molecular functions. IPA software considered cellular and molecular functions with a regulation Z-score value of ≥2.0 to be significantly increased and cellular and molecular functions with a regulation Z-score value of ≤ − 2.0 to be significantly decreased. These genes and their corresponding RNA-Seq fold changes were further analysed for enriched KEGG pathways and GO terms using the “EnrichDAVID” function in ClusterProfiler. Pathways and GO terms from the DAVID ClusterProfiler analyses were considered enriched at a P-value of less than 0.05 and an FDR of 5%.

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