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Background  Eggshell formation takes place in the shell gland of the oviduct of laying hens. The eggshell is rich in calcium and various glycoproteins synthesised in the shell gland. Although studies have identified genes involved in eggshell formation, little is known about the regulation of genes in the shell gland particularly in a temporal manner. The current study investigated the global gene expression profile of the shell gland of laying hens at different time-points of eggshell formation using RNA-Sequencing (RNA-Seq) analysis.    Results  Gene expression profiles of the shell gland tissue at 5 and 15 h time-points were clearly distinct from each other. Out of the 14,334 genes assessed for differential expression in the shell gland tissue, 278 genes were significantly down-regulated (log 2  fold change > 1.5; FDR < 0.05) and 413 genes were significantly up-regulated at 15 h relative to the 5 h time-point of eggshell formation. The down-regulated genes annotated to Gene Ontology (GO) terms showed anion transport, synaptic vesicle localisation, organic anion transport, secretion and signal release as the five most enriched terms. The up-regulated gene annotation showed regulation of phospholipase activities, alanine transport, transmembrane receptor protein tyrosine kinase signalling pathway, regulation of blood vessels diameter and 3, 5-cyclic nucleotide phosphodiesterase activity as the five most enriched GO terms. The putative functions of genes identified ranged from calcium binding to receptor activity. Validation of RNA-Seq results through qPCR showed a positive correlation.    Conclusions  The down-regulated genes at 15 h relative to the 5 h time-point were most likely involved in the transport of molecules and synthesis activities, initiating the formation of the eggshell. The up-regulated genes were most likely involved in calcium transportation, as well as synthesis and secretory activities of ions and molecules, reflecting the peak stage of eggshell formation. The findings in the current study improve our understanding of eggshell formation at the molecular level and provide a foundation for further studies of mRNA and possibly microRNA regulation involved in eggshell formation in the shell gland of laying hens.    Electronic supplementary material  The online version of this article (10.1186/s12864-019-5460-4) contains supplementary material, which is available to authorized users.

RNA-sequencing analysis of shell gland shows differences in gene expression profile at two time-points of eggshell formation in laying chickens