GSM8895201: sham_7352_liver; Mus musculus; RNA-Seq
Single nuclear suspensions were produced in three sham and three SBR samples according to the protocol outlined by Nadelmann et. al. (Nadelmann, E. R. et al. Isolation of Nuclei from Mammalian Cells and Tissues for Single‐Nucleus Molecular Profiling. Current Protocols 1, e132 (2021).) In brief, liver tissue was gently homogenized to release cells. Lysate was added to lyse cells and release nuclei. The nuclei were filtered and stained with NucBlue. They were then sorted with FACS. Single nuclear suspension concentrations were estimated using Countess. cDNA was prepared after the GEM generation and barcoding, followed by the GEM-RT reaction and bead cleanup steps.  Purified cDNA was amplified for 11-13 cycles before being cleaned up using SPRIselect beads.  Samples were then run on a Bioanalyzer to determine the cDNA concentration.  GEX libraries were prepared as recommended by the 10x Genomics Chromium Single Cell 3' Reagent Kits User Guide (v3.1 Chemistry Dual Index) with appropriate modifications to the PCR cycles based on the calculated cDNA concentration.  For sample preparation on the 10x Genomics platform, the Chromium Next GEM Single Cell 3' Kit v3.1, 16 rxns (PN-1000268), Chromium Next GEM Chip G Single Cell Kit, 48 rxns (PN-1000120), and Dual Index Kit TT Set A, 96 rxns (PN-1000215) were used.  The concentration of each library was accurately determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer's protocol (KAPA Biosystems/Roche) to produce cluster counts appropriate for the Illumina NovaSeqX instrument.  Normalized libraries were sequenced on a NovaSeqX Flow Cell using the 151x10x10x151 sequencing recipe according to manufacturer protocol.  Read 1 was trimmed to the 10x Genomics recommendation of 28bp. A median sequencing depth of 50,000 reads/cell was targeted for each Gene Expression Library.