Single-nucleus RNA-seq of adult chimpanzee and bonobo livers
Single-nucleus RNA-seq of adult chimpanzee and bonobo livers
Livers were dissected from adult animals after euthanasia, cut into small pieces, and flash-frozen in a 1.5 or 2 mL Eppendorf tube in liquid nitrogen. RNA was extracted from tissue pieces for quality control. About 5-10 mg of liver tissue was used for each nuclei extraction. Each sample was homogenized with a micropestle in 400 μl ice-cold homogenization buffer (250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris-HCl (pH 8), 0.1% IGEPAL, 1 μM DTT, 0.4 U/μl Murine RNase Inhibitor (New England BioLabs, cat# M0314L), 0.2 U/μl SUPERas-In (Ambion, cat# AM2694), and cOmplete Protease Inhibitor Coctail (Roche, cat# 11 836 145 001)). The homogenates were triturated gently by a P1000 tip for 10 times, incubated on ice for 5-10 minutes, and then centrifuged at 100g for 1 minute at 4 °C to pellet any unlysed tissue chunks. The supernatant was transferred into another 1.5 mL Eppendorf tube and centrifuged at 400g for 4 minutes at 4 °C to collect nuclei. The nuclei were washed twice in 400 μl homogenization buffer and strained by a 40 μm Flowmi strainer (Sigma, BAH136800040) during the second wash step to remove nuclei aggregates. The final nuclei pellet was resuspended in 30-50 μl Nuclei buffer (10X Genomics, PN-2000207). To estimate the nuclei concentration, nuclei aliquots were diluted in PBS with Hoechst and PI DNA dyes and counted on Countess II FL Automated Cell Counter (Thermo Fisher Scientific). Around 15,000 -20,000 nuclei were used as input for the single-nuclei RNA-seq experiment. The Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 were used for generating the single-nuclei RNA-seq libraries following the manufacturer's instructions. Libraries were quantified on a Qubit Fluorometer (Thermo Fisher Scientific) and the profiles were assessed on a Fragment Analyzer (Agilent) for quality control. 