GSM5015779: LV22: HFD, biological replicate 10; Mus musculus; RNA-Seq
Liver were removed, flash frozen in liquid N2 and stored at -80C until day of RNA isolation; total RNA was harvested using Direct-zol RNA Miniprep Plus kit (Zymo Research). RNA purity and integrity were evaluated using a Bioanalyzer RNA 6000 Nano chip (Agilent Technologies). Lexogen QuantSeq 3'mRNA-Seq Library Prep Kit-FWD for Illumina sequencing was used with 325 ng of total RNA for the construction of sequencing libraries. Library preparation was started by oligo(dT) priming, with primers already containing the Illumina-compatible linker sequence for Read 2. After firststrand synthesis, the RNA was removed before random primers that contained the corresponding Illumina-compatible linker sequence for Read 1 initiated the second-strand synthesis. Second strand synthesis was followed by a magnetic bead-based purification step. The libraries were PCR amplified introducing sequences required for cluster generation and i7 and i5 dual indices (Lexogen i7 6 nt Index Set and Lexogen i5 6 nt Unique Dual Indexing Add-on Kit) for 16-20 PCR cycles with the optimal number predetermined by qPCR with the PCR Add-on Kit for Illumina (Lexogen GmbH). After a second magnetic bead-based purification, libraries were quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and sized using an Agilent High Sensitive D5000 Screen Tape (Agilent Technologies) to determine molarity. Equal molar amounts of the libraries were multiplexed and then sequenced on an Illumina Hiseq3000 platform (Illumina, San Diego, CA, USA) at the Center for Genome Research and Biocomputing, Oregon State University using single-end sequencing with 100-bp reads.