Dnasei kit

Total RNA from cell lines and mice tissues was isolated using Trizol reagent (Invitrogen, Carlsbad, CA USA). RNA was treated with DNaseI using the DNaseI Kit (Ambion, Ambion Inc., Austin, TX, USA). cDNA was synthesized from 0.5–1 μg total RNA using the Quanta Biosciences qScript ™ (Quanta Biosciences Inc., Gaithersburg, MD, USA) cDNA Synthesis Kit (95047-100) for mRNA analysis and the qScript™ microRNA cDNA Synthesis Kit (95107-100) for miRNA analysis. Quantiative PCR (qPCR) of miRNAs and mRNAs in RNA isolated from cultured cell or mice livers was performed using the CFX384, C1000 touch thermal cycler (Bio-Rad, Hercoles, CA, USA) and a SYBR Green PCR Kit: Quanta Cat. #84018 and #84071, respectively. All experiments were performed in triplicate. qRT-PCR of RNA isolated from human samples was performed using the following specific TaqMan (TAqMan, Invitrogen, CA, USA) predesigned probes: Hs00399294_g1 (H19),Hs04187499_m1 (FADD), and hsa-miR-675-002005 (miR-675).

RNA was isolated by cesium centrifugation through 2 ml of 5.7M cesium chloride at 39000 rpm at 20°C for 18 hours and DNAse treated with DNAse 1 from the Ambion DNAse I Kit. 1 μg of RNA was used from each sample for library preparation with the TrueSeq Stranded Total RNA kit from Illumina. Ribosomal RNA was depleted using rRNA binding beads and the RNA was fragmented and primed with random hexamers. First strand synthesis was done using a mixture containing Superscript II and Actinomycin D to synthesize RNA dependent DNA and inhibit DNA dependent RNA respectively. Second strand synthesis was done using dUTP instead of dTTP to create stranded cDNA. Libraries were then adenylated at the 3’ end and adaptors containing identifier sequences and flow-cell binding sequences were ligated to both ends. Finally the cDNA fragments were amplified for 12 cycles instead of 15. The Agilent Technologies 2200 Tapestation was used to verify library size at ~260bp. Sequencing was done on a HiSeq 2000 by Perkin Elmer.