MCMV infection of newborns severely compromised NK cell phenotype, maturation and functionality. Inflammatory responses to infection dysregulated the expression of major transcription factors governing NK cell fate.
RNA sample preparation, quality control and sequencing. To determine changes in NK cells in the postnatal period, splenocytes were isolated from spleens of naive mice at postnatal days 7, 14, 21 and 60. To determine the impact of infection on NK cells, splenocytes were isolated from spleens of naive or MCMV-infected mice at day 21 p.i. Following isolation, splenocytes were labeled with anti-CD45, anti-CD3, anti-CD19, anti-NK1.1, and anti-DX5 antibodies, and NK cells defined as CD45+, CD3-, CD19-, NK1.1+, and DX5+ cells, were separated from the mixture using fluorescence-activated cell sorting on FACSAriaIIu using a 70-µm nozzle.  Sort purity was determined by sorting an aliquot of cells into 10% RPMI and then immediately reanalyzing the sorted aliquot by flow cytometry. In general, we achieved sort purities of > 98%. NK cells for RNA isolation were sorted directly into RLT lysis buffer (Qiagen) and their total RNA was isolated using RNeasy Micro Kit (Qiagen), according to the manufacturer's recommendations.