GSM7681268: Isolated hepatocytes Senescent, rep 2; Mus musculus; RNA-Seq
Hepatocytes were isolated from 10 – 12 week old mice following the standard two-step collagenase perfusion technique. Briefly, mice received a terminal dose of anesthetic and underwent laparotomy. The portal vein was cannulated and then perfused with 30 mL Liver Perfusion Medium (Gibco) followed by 50 mL Liver Digest Medium (Gibco) pre-warmed to 37°C. The liver was removed and then hepatocytes were mechanically disassociated and released from the Glisson capsule, filtered through 70 um filter and purified with a density gradient centrifugation. 500ng of each total-RNA sample was used for RNA-seq library preparation, using the NEBNEXT Ultra II Directional RNA Library Prep Kit (NEB #7760) and the Poly-A mRNA magnetic isolation module (NEB #E7490) according to the manufacturer's protocol. AMPure XP beads (Beckman Coulter #A63881) were used to purify the cDNA library. Multiple indexing adapters were ligated to the ends of the ds cDNA to prepare them for hybridisation onto a flow cell, before 10 cycles of PCR were used to selectively enrich those DNA fragments that had adapter molecules on both ends and amplify the amount of DNA in the library suitable for sequencing. Following amplification, libraries were purified using AMPure XP beads. Completed libraries were quantified by fluorometry using the Qubit dsDNA HS assay and assessed for quality and fragment size using the Agilent Bioanalyser with the DNA HS Kit (#5067-4626).