GSM9156002: LCB1-V1; Mus musculus; RNA-Seq
Total RNA from mouse liver was extracted using TRIzol (Sigma) and quality control-confirmed RNA samples were submitted to GENEWIZ/Azenta (South Plainfield, NJ) for Standard RNA-Seq using Next Generation Sequencing, or to quantify gene expression using nCounter Mouse Metabolic Pathways panel. RNA quality control, library preparations, sequencing reactions and initial bioinformatic analysis were conducted at GENEWIZ, LLC (South Plainfield, NJ, USA). RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked using Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA). Only samples with integrity number ≥7 were used. RNA-sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina using manufacturer's instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were first enriched with Oligo(dT) beads. Enriched mRNAs were fragmented for 15 min at 94°C. First strand and second strand cDNAs were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3'ends and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by limited-cycle PCR. The sequencing libraries were validated on the Agilent TapeStation and quantified by using Qubit 2.0 Fluorometer as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). The sequencing libraries were clustered on flow-cells.