title: GSM6745647: 10x snRNA-seq on the mouse liver (922e80); Mus musculus; RNA-Seq
library_construction_protocol: The liver nuclei were isolated following the protocol described by Thrupp et al., 2020. For the fresh samples, 200 mg of fresh mouse liver tissue was minced and transferred to a Dounce homogenizer cylinder containing 1 mL of ice-cold homogenization buffer (320 mM Sucrose, 5 mM CaCl2, 3 mM Magnesium Acetate, 10 mM Tris-HCl pH 7.5, 0.1 mM EDTA, 0.1% Igepal, 0.1 mM PMSF, 1 mM βME and 0.2 U/µL RNasin Plus RNase Inhibitor (Promega). For the frozen samples, a piece of 200 mg of liver tissue was sectioned on dry ice and transferred to a Dounce homogenizer cylinder containing 1 mL of ice-cold homogenization buffer and let to thaw for 5 minutes. From this step onward, both the fresh and frozen tissue were homogenized with 10 strokes of pestle A and 10 strokes of pestle B until a homogeneous nuclei suspension was achieved. The resulting homogenate was filtered through a 70-μm cell strainer (Corning). Next, we added 1.65 mL of homogenization buffer to top the homogenate up and mixed it with 2.65 mL of gradient medium (5 mM CaCl2, 50% Optiprep, 3 mM Magnesium Acetate, 10 mM Tris, 0.1mM PMSF, 1mM βME). 4 mL of 29% Iodoxanol cushion was prepared with a diluent medium (250 mM sucrose, 150 mM Potassium chloride, 30 mM Magnesium chloride, 60 mM Tris pH 8) and added into an ultracentrifuge tube. Following, 5.3 mL of sample (mixed with the homogenization buffer and the gradient medium) was gently layered on top of the 29% Iodoxanol cushion. Samples were centrifuged in a SW41Ti rotor at 7700 x g, 4°C for 30 min and the supernatant was gently removed without disturbing the nuclei pellet. Nuclei were resuspended in 200 μL of resuspension buffer (1x PBS, 1% BSA and 0.2 U/μL Rnasin Plus RNase Inhibitor (Promega)) and transferred to a 1.5 mL Eppendorf tube. 9 μL of sample was mixed with 1 μL of arginine orange/propidium iodide (AO/PI) stain, loaded onto a LUNA-FL slide and visualized with the LUNA-FL Automated cell counter for nuclei yield, morphology, and presence of clumps/debris. Single-nuclei libraries were generated using the 10x Chromium Single-Cell Instrument and Chromium Single Cell 3' Reagent v3 Kits (10x Genomics) according to the manufacturer's protocol. In brief, the single mouse liver nuclei suspension was loaded in the Chromium chip B for partitioning into nanoliter-scale Gel Beads-in-emulsion (GEMs). After GEMs generation, the obtained emulsion was incubated in a C1000 Touch Thermal Cycler (Bio-Rad) under the following program: 53°C for 45 min, 85°C for 5 min and hold at 4°C. Incubation of the GEMs produced barcoded, full-length cDNA from poly-adenylated mRNA. After incubation, single-cell droplets were dissolved, and full-length cDNA were isolated using Cleanup Mix containing Silane Dynabeads. To generate sufficient mass for library construction, the cDNA was amplified via PCR: 98°C for 3 min; 12 cycles of 98°C for 15 s, 63°C for 20 s, 72°C for 1 min; 72°C for 1 min; and hold at 4°C. Subsequently, the amplified cDNA was fragmented, end- repaired, A-tailed and index adaptor ligated, with SPRIselect cleanup in between steps. The final gene expression library was amplified by PCR: 98°C for 45 s; 10-12 cycles of 98°C for 20 s, 54°C for 30 s, 72°C for 20 s. 72°C for 1 min; and hold at 4°C. The sequencing-ready libraries were cleaned up with SPRIselect beads. Before sequencing, the fragment size of every library was analyzed using the Bioanalyzer high-sensitivity chip and were sequenced on HiSeq2500 or NovaSeq6000 instruments with the following sequencing parameters: 28 bp read 1 – 10 bp index 1 (i7) – 10 bp index 2 (i5) – 75 bp read 2.