RNA-seq analysis of Vitamin K2 treated WT worms

We used RNA-seq analysis to identify changes in gene expression occurring in the worms treated with VK2, and based on these sequencing data, more than fifteen hundred genes were identified as up-regulated by as much as 1.5 times subsequent to VK2 treatment. These differentially expressed genes were analyzed for pathway and gene ontology (GO) enrichment, which indicated that the genes influenced by VK2 treatment were enriched in several significant signaling pathways, including longevity, stress resistance, peroxisome, and fat metabolism pathways. Overall design: Synchronized young adults were cultured on NGM plates with or without 0.1 µM VK2 and incubated at 20°C for seven days. The worms were transferred to new NGM plates with or without VK2 every day to get rid of the progeny and then collected. Total RNA quality was measured using a NanoDrop ND-1000 (Agilent Technologies). Library preparation was performed using a KAPA Stranded RNA-Seq Library Prep Kit (Illumina), and the quality was confirmed using an Agilent 2100 Bioanalyzer (Agilent Technologies). Paired-end sequencing was performed on an Illumina NovaSeq 6000. Transcriptome alignment and quantification were performed using Hisat2 software. C. elegans genome version WBcel235 was used as the reference. Differentially expressed genes were identified using the Ballgown package of R project to calculate the Fragments Per Kilobase of gene/transcript model per million mapped fragments (RPKM). The newest version of KEGG pathway enrichment and Gene Ontology were performed using R project or Python developed by Aksomics Inc. The heat maps were generated using Group FPKM (The average of log scaled FPKM: log2(FPKM + 1) of the genes in the groups).