A Human Milk Oligosaccharide 3-Fucosyllactose Primes Cellular Viral Defense by Coordinately Modulating Interferon Receptors and Interferon-Stimulated Genes

The Calu-3 cell lines were cultured in PBS- or 2-Fucosyllactose- or 3-Fucosyllactose-supplemented media for 3 passages at 37 celsius degree in 5 percent CO2. Then the cultured cells were harvested in TRIZOL reagents for analysis. The libraries were prepared for 151bp paired-end sequencing using TruSeq stranded mRNA Sample Preparation Kit (Illumina, CA, USA). Namely, mRNA molecules were purified and fragmented from 1 microgram of total RNA using oligo (dT) magnetic beads. The fragmented mRNAs were synthesized as single-stranded cDNAs through random hexamer priming. By applying this as a template for second strand synthesis, double-stranded cDNA was prepared. After sequential process of end repair, A-tailing and adapter ligation, cDNA libraries were amplified with PCR (Polymerase Chain Reaction). Quality of these cDNA libraries was evaluated with the Agilent 2100 BioAnalyzer (Agilent, CA, USA). They were quantified with the KAPA library quantification kit (Kapa Biosystems, MA, USA) according to the manufacturer's library quantification protocol. Following cluster amplification of denatured templates, sequencing was progressed as paired-end (2x151bp) using Illumina NovaSeq6000 (Illumina, CA, USA)