Next Generation Sequencing and Analysis of the Transcriptomes of Wild-type and Alkbh5-/- Neutrophils

By performing transcriptome-wide RNA sequencing (RNA-seq) analysis on the peritoneal neutrophils from Alkbh5-deficient mice (Alkbh5-/-) and Wild-type littermates (Alkbh5+/+) at 12h or 36h after mild cecal ligation and puncture (CLP), respectively, we want to identify potential targets of ALKBH5 and characterize the transcriptional landscape in neutrophils during antibacterial innate defense. Gene Ontology biological processes enrichment analysis of the significantly differentially expressed genes (DEGs) showed that neutrophil migration made up the most significantly enriched biological processes with annotations of neutrophil association upon loss of ALKBH5 in neutrophils, at both 12h and 36h after CLP. Many significantly DEGs also encompassed transcriptional signatures related to neutrophils, specifically to neutrophil influx into the infection site, including chemotaxis, response to chemokine, extravasation, ERK1 and ERK2 cascade, homeostasis of neutrophils. ALKBH5 deletion led to significantly decreased transcript expression of neutrophil migration-promoting Cxcr2 and Nlrp12; while increased transcript expression of neutrophil migration-suppressive Ptger4, Tnc, and Wnk1. These results demonstrated that ALKBH5 imprints migration-promoting transcriptional landscape in neutrophils to enable their migration into the site of infection for antibacterial innate defense. Transcriptomes profiles of gene expression changes in peritoneal neutrophils from Alkbh5-deficient mice (Alkbh5-/-) and Wild-type littermates (Alkbh5+/+) at 12h or 36h after mild CLP, respectively. Four biological replicates.