P66shc KO reprograms hepatic transcriptome oscillations

Eight- to ten-week-old male P66Shc KO mice and their littermate control mice were entrained to a 12-h LD cycle for at least 10 days and then transferred to complete darkness. After dark adaptation, six mice (three WT and three for p66Shc KO) were sacri?ced every 4 h for 24 h under dim red light, and livers were harvested and stored at -80°C until use in the RNA-seq analysis. Total mRNA was extracted from all three mice at each time point, pooled and then divided into two samples. Twenty-four samples were then sent for sequencing. A total amount of 2 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (#E7530L, NEB, USA) following the manufacturer's recommendations and index codes were added to attribute sequences to each sample. The clustering of the index-coded samples was performed on a cBot cluster generation system using HiSeq PE Cluster Kit v4-cBot-HS (Illumina) according to the manufacturer's instructions. After cluster generation, the libraries were sequenced on an Illumina Hiseq 4000 platform and 150 bp paired-end reads were generated. The transcript abundance is expressed as FPKM (fragments per kilobase of exon model per million mapped reads) values.