1.0 mm zirconia beads

Plasma samples and resected liver tissue samples were analyzed for BAs using UPLC-MS as described [43 (link)]. In brief, plasma samples were prepared for analysis using protein precipitation with methanol, and liver samples were lyophilized by 24-h freeze drying. BAs were extracted from dried liver samples using a mixture containing water, acetonitrile, and 2-propanol in a 2:1:1 volume ratio. Samples were pulverized in a Biospec bead beater with 1.0-mm Zirconia beads and centrifuged at 16,000× g for 20 min at 4 °C. Quality control samples were prepared from equal parts of plasma supernatant and were used to monitor the performance of the assay. To determine chromatographic retention times of BAs, mixtures of BA reference standards were analyzed following the analysis of study samples. Plasma and liver BA analysis was performed as described [43 (link)]. Relative BA intensities were corrected for the liver sample’s dry weight.

Total RNA was extracted from embryos using a Beadbeater (Biospec, Cat. #607) with 1.0 mm zirconia beads (Biospec, #11079110zx) and the RNAdvance Tissue kit (Agencourt #A32649) according to the manufacturer’s instructions, including DNaseI treatment. We systematically checked on a Bioanalyzer RNA Nano chip (Agilent) that the RNA was of very high quality. Libraries were prepared as described before (Batut et al., 2013 (link); Batut and Gingeras, 2013 (link)). 5’-monophosphate transcripts were depleted by TEX digest (Epicentre #TER51020). For every time series, each sample was labeled with a different sequence barcode during reverse-transcription, and all samples for the series were then pooled and processed together as a single library. Quality control and library quantification were carried out on a Bioanalyzer DNA High Sensitivity chip. Each library was sequenced on one lane of an Illumina HiSeq 2000.

Muscle tissue which was flash frozen and stored at −80 °C was retrieved and a ~20 mg portion was homogenized with a Minin-BeadBeater 8 (Biospec) while in a vial containing 1 ml of TRIzol and 1.0 mm zirconia beads (BioSpec, Cat#11079110zx). RNA was isolated using the RNAqueous Phenol-free total RNA Isolation Kit (Ambion, Cat# AM1912). cDNA was synthesized using the RT² First Strand Kit (Qiagen, Cat# 330401). Samples were then analyzed using the Mouse Skeletal Muscle: Myogenesis & Myopathy RT² Profiler™ PCR Array (Qiagen, Cat# PAMM-099Z) and RT² SYBR Green Mastermix (Qiagen, Cat# 330523) per manufacturer’s instructions with use of an Applied Biosystems 7500 Real-Time PCR instrument. Fold changes and P values were determined from C_t values analyzed using the analysis template provided by Qiagen (PCRArrayDataanalysis_V4). Criteria for gene expression exceeding 1.3-fold change and P < 0.05 were used to identify differentially expressed genes. Bioinformatic analysis was performed for differentially expressed genes using Ingenuity Pathway Analysis (IPA; Ingenuity Systems, www.ingenuity.com). IPA biological functions with the exception of cancer specific functions were evaluated.