Cataloguing the postnatal small intestinal transcriptome during the period of susceptibility to necrotizing enterocolitis

In the first postnatal month, the developing mouse intestine shifts from an immature to a mature intestine that will sustain the organism throughout the lifespan. Here, we surveyed the mouse intestine in C57Bl/6 mice by RNA-Seq to evaluate the changes in gene expression over time from the day of birth through 1 month of age in both the duodenum and ileum. We analyzed gene expression for changes in gene families that correlated with the periods of NEC susceptibility or resistance. We highlight that increased expression of DNA processing genes and vacuolar structure genes, tissue development and morphogenesis genes, and cell migration genes all correlated with NEC susceptibility, while increases in immunity gene sets, intracellular transport genes, ATP production, and intracellular metabolism genes correlated with NEC resistance. Using trends identified in the RNA analyses, we further evaluated expression of cellular markers and epithelial regulators, immune cell markers, and adenosine metabolism components. We confirmed key changes with qRT-PCR and immunofluorescence. In addition, we compared some findings to humans using human intestinal biopsies and organoids. This dataset can serve as a reference for other groups considering the role of single molecules or molecular families in early intestinal and postnatal development. Overall design: Small intestinal samples were collected from 0 day, 2d, 4d, 6d, 8d, 2 week, 3wk, 4wk old C57Bl6/J mice. For the purposes of RNA-Seq, 2cm of proximal intestine immediately after the pylorus was collected for the “duodenum” sample and 2cm of distal intestine immediately before the ileocecal junction was collected for the “ileum” sample. The library was prepared by the Dana Farber Next Generation Sequencing Core Facility using a KAPA library quantification kit (Roche). Sequencing was done on an Illumina NovaSeq 6000 with total RNA.