title: NextSeq 550 sequencing; Single-Nucleus RNA Sequencing Identifies New Classes of Proximal Tubular Epithelial Cells in Kidney Fibrosis
design: Single-Nucleus RNA Sequencing Identifies New Classes of Proximal Tubular Epithelial Cells in Kidney Fibrosis
library_construction_protocol: The experiment was carried out by using kidneys harvested from 4 AAN mice and 4 naive control. One quarter of a harvested kidney from each mouse was minced into <2 mm pieces and transferred to a Dounce tissue grinder containing 2 ml of lysis buffer (Nuclei EZ Lysis buffer (Sigma NUC101) supplemented with protease inhibitor (Sigma 5892970001) and RNase inhibitors (Promega N2615 and Life Technologies AM2696)). Each kidney sample was homogenized and transferred into 50 ml tubes containing 2 ml of lysis buffer, incubated for 7 min on ice, filtered through a 40 μm cell strainer, and centrifuged at 500g for 5 min at 4oC. The pellet was resuspended in 4 ml lysis buffer, incubated for another 7 min on ice, then centrifuged again at 500g for 5 min at 4oC. The pellet was then resuspended in 4 ml wash & resuspension buffer (1xPBS, 1.0% BSA, and RNase Inhibitor (Sigma 3335399001)) and filtered through a 20 μm cell strainer. Samples were then processed immediately using the 10x Genomics single-cell library preparation protocol. Library preparation was performed using Chromium Single Cell 3ʹ Reagent Kits v3 (10x Genomics) and cDNA quality was evaluated by fragment analysis (5200 Fragment Analyzer System, Agilent). Library preparation was performed using Chromium Single Cell 3ʹ Reagent Kits v3 (10x Genomics) and cDNA quality was evaluated by fragment analysis (5200 Fragment Analyzer System, Agilent).