GSM9393541: snRNA-seq tdtom CTRL; Mus musculus; RNA-Seq
Single lobes were isolated from mouse liver and snap frozen in liquid nitrogen. On the day of nuclei isolation, snap frozen samples were gently split with a razor blade to yield small chunks of approximately 8-10mm in diameter. Homogenization buffer (HB) was prepared as described95. The sample was placed in a 1.5mL tube with 750uL homogenization buffer (0.1% Triton-X, 1 M dithiothreitol, 242.25 mM sucrose, 24.23 mM KCl, 4.85 mM MgCl2, 9.69 mM Tris-pH 8.0) then dounced 5 times with a loose pestle and 10 times with a tight pestle. The sample was then transferred to a fresh tube and 250 L of HB was added before incubation on ice for 5 minutes. The homogenized sample was then filtered through a 100uM cell strainer (Corning, 08-771-19). Filtrate was then passed through the 35 M strainer cap of a polystyrene test tube (Corning, 08-771-23). The sample was spun down at 500 x g for 5 minutes at 4C. Density gradient purification was performed with Optiprep Density Gradient Medium (Sigma-Aldrich, 92339-11-2) as described in Narayanan et al. 2020. Isolated nuclei were stained in a 1:1 ratio with 0.4% Trypan Blue solution and observed in a hemocytometer under the Echo Rebel Microscope. Samples were fixed and permeabilized using the Parse Biosciences Evercode™ Fixation kit v2 (ECF2103) according to manufacturer instructions. Samples were then stored at –80C until needed for library preparation. Libraries were prepared using Parse Biosciences Evercode™ Whole Transcriptome v2 barcoding kit according to manufacturer instructions as described in Evercode™ user manual v2.2.2. Libraries were sequenced at the Yale Center for Genome Analysis on the NovaSeqX, 100bp paired-end sequencing.