GSM6032161: Liver, TAA, 3 weeks, replicate 3; Mus musculus; RNA-Seq
Hepatic nuclei were isolated from flash frozen liver chunks. Briefly, tissue samples were cut into pieces of approximately 25 mg and immediately homogenized using a Kimble Dounce tissue grinder (Sigma-Aldrich, D8938) by performing 15 strokes with pestle A in 2 mL of ice-cold nuclei lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, and 0.1% IGEPAL ® CA-630). Then, another 2 mL of nuclei lysis buffer were added to each sample and lysis proceeded on ice for 10 min followed by straining the lysates through 40 µm cell strainers (Falcon, Corning). Lysates were centrifuged at 500 g for 5 min at 4°C and resuspended in 4 mL of nuclei wash buffer (PBS supplemented with 1% BSA and 0.2U/µL RNasin® Plus Ribonuclease Inhibitor (Promega, N2615). Following another round of centrifugation, nuclei were resuspended in 700 µL of nuclei wash buffer, stained with 4',6-Diamidine-2'-phenylindole dihydrochloride (DAPI) at 0.1 µg/mL and propidium iodide (PI) at 2.5 µg/mL. Nuclei preparations were strained through 70 µm cell strainers prior to fluorescence-activated cell sorting (FACS) sorting with a two-laser configuration (488nm 80mW and 640nm 50mW) BD FACSJazz™ stream-in-air cell sorter (BD Biosciences), equipped with a 100 µm nozzle operating at a sheath pressure of 27 psi. Machine calibration was performed by flow cytometry facility staff before each sort using Sphero™ 8-peak rainbow calibration beads (BioLegend) to achieve optimal stream alignment, laser alignment and target mean fluorescence intensities for each detector. The drop delay was determined by setting a value that resulted in total side-stream deflection of Accudrop Beads (BD Biosciences) sorted through a 640nm 5mW laser that bisected center and side streams. Nuclei were identified through an initial FSC-H/SSC-H gate, followed by the discrimination of single events exhibiting proportional FSC-W/FSC-H profiles. Within single events, nuclei were identified as PI positive events. Two peaks of PI-positive events were visualized corresponding to 2n and 4n nuclei; both peaks were pooled together for sorting and downstream 10x Genomics analysis to maintain hepatic cell representation unaltered. 50,000 PI-positive events were sorted per sample, then post-sort nuclei concentration and quality were checked using a fluorescence microscope and hemocytometer. For the construction of snRNA-seq libraries, 10x Genomics Chromium Single Cell 3'v3 Reagent Kits were used according to manufacturer's instructions. 10,000 freshly sorted nuclei were loaded onto a 10x Genomics Chromium Single Cell 3ʹ v3 chip B and processed immediately in a 10x Chromium controller. Specifically, we utilized 19 PCR cycles for cDNA amplification. Libraries were quantified with qPCR using the NEBnext Library Quant Kit for Illumina and fragment size assessed with TapeStation D1000 kit (Agilent). Libraries were pooled in equimolar concentration and sequenced using an Illumina NovaSeq 6000 and S2 flow cells (100 cycle kit) with a read one length of 28 cycles, and a read two length of 94 cycles. snRNA-seq