Hepatocytes were resuspended in TRIzol (Thermo Fischer 15596026) and stored at -20°C until the day of library preparation, when RNA was isolated from TRIzol per the manufacturer's protocol and brought through at least cDNA synthesis all on the same day. RNA isolated from TRIzol was resuspended in TE. Next, total RNA was polyA-selected using 50 μL Invitrogen Dynabeads (dT)25-61002 per the manufacturer's protocol. Sequencing libraries were prepped from polyA+ RNA using the NEBNext Ultra Directional Library Prep Kit for Illumina (NEB 7420), with 12 cycles of PCR using NEBNext Oligos. After library prep, library size was assessed using a Bioanalyzer and Agilent DNA 1000 chip (Agilent 5067-1504), and the concentration of amplifiable DNA assessed with the NEBNext Library Quantification Kit (NEB E7630). Individual libraries were diluted to 2 nM, pooled, and the pooled library requantified with the NEBNext Next Library Quantification Kit. The pooled library was diluted to 3.2 pM using the NEBNext 500/550 High Output Reagent Cartridge V2 75 cycles (Ref 15057934) and loaded onto the NextSeq 500 for 75bp single-end sequencing. NEBNext Ultra Directional Library Prep Kit for Illumina (NEB 7420)
GEO Accession
GSM3346526