GSM9725562: 6100.23947.2233916.S51.W104502; Mus musculus; RNA-Seq
Total RNA was purified from liver. Tissue samples were transferred from RNAlater (ThermoFisher, Cat # AM7021) to 1-3 milliliters Trizol reagent (ThermoFisher, Cat # 15596026). Samples are homogenized on a custom Omni homogenizer (Omni, Cat #SKU: 51-000-1) at 20,000 RPM for 180 seconds. Lysates were phase separated with chloroform and the aqueous phase was purified on the Kingfisher flex (ThermoFisher, Cat # 5400630) with the MagMAX-96 for Microarrays Total RNA Isolation Kit (ThermoFisher, Cat # AM1839) with an additional DNAse (Qiagen, Cat # 79254) step added between the first and second washes. RNA is quantified on the Lunatic (Unchained Labs, Cat # 700-2000) using a standard Lunatic plate (Unchained Labs, Cat # 701-2019), and integrity was read on the 5300 Fragment Analyzer (Agilent, Cat #  M5311AA) with the RNA Kit (Agilent, Cat # DNF-471-1000) according to manufactures protocol. mRNA-seq libraries were generated using KAPA® mRNA HyperPrep Kit (Roche Sequencing). Starting material was 500 ng RNA and fragmentation was done at 85°C for 6 minutes. cDNA was ligated with 1.5 µM xGen Dual Index UMI Adapters (Integrated DNA Technologies) and amplified using 12 PCR cycles. Sequencing of the resulting libraries was done on NovaSeq 6000 (Illumina) using a 51 cycle, single-end sequencing recipe.