GSM8482483: liver, Sham, rep3; Mus musculus; RNA-Seq
Nucleus isolation of tissue samples was done as previously described. Briefly, tissue samples were cut into pieces <0.5 cm and homogenized using a glass Dounce tissue grinder (Sigma, cat. no. D8938). The tissue was homogenized 25 times with pestle A and 25 times with pestle B in 2 ml of ice-cold nuclei EZ lysis buffer. The sample was then incubated on ice for 5 min, with an additional 3 ml of cold EZ lysis buffer. Nuclei were centrifuged at 500g for 5 min at 4 °C, washed with 5 ml ice-cold EZ lysis buffer and incubated on ice for 5 min. After centrifugation, the nucleus pellet was washed with a 5 ml nuclei suspension buffer (NSB; consisting of 1× PBS, 0.01% BSA and 0.1% RNase inhibitor (Clontech, cat. no. 2313A)). Isolated nuclei were resuspended in 2 ml NSB, filtered through a 35 μm cell strainer (Corning-Falcon, cat. no. 352235) and counted. A final concentration of 1,000 nuclei per μl was used for loading on a 10x channel. We prepared single-nucleus RNA-seq libraries with Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 on the Chromium Controller (10× Genomics). We prepared single nucleus suspensions from cultured cell lines and nuclei were suspended in PBS containing 0.04% BSA. The nuclei suspension was loaded onto the Chromium Next GEM Chip G and ran the Chromium Controller to generate single-cell gel beads in the emulsion (GEMs) according to the manufacturer's recommendation. Captured nuclei were lysed and the released RNA was barcoded through reverse transcription in individual GEMs. Barcoded, full-length cDNA was generated and libraries were constructed according to the performer's protocol. The quality of libraries was assessed by Qubit 4.0 and the Agilent 2100. Sequencing was performed on the Illumina NovaSeq 6000 with a sequencing depth of at least 50,000 reads per nucleus and 150 bp (PE150) paired-end reads (performed by Biomarker Technologies Corporation, Beijing, China).