title: GSM8217478: C4,control,replicate3,snRNA-seq; Homo sapiens; RNA-Seq
library_construction_protocol: Single nuclei were isolated from fresh or liquid nitrogen flash frozen renal biopsies using the 10X Genomics Chromium Nuclei Isolation Kit with RNase Inhibitor 16 rxns (PN-1000494), with protocol modifications includingsupplementedRNase inhibitor and FACS sorting. The day prior to the nuclei isolation, polypropylene 1.5ml Eppendorf tubes were coated with 10% BSA (MACS BSA Stock Solution, Miltenyi Biotec, 130-091-376) overnight at 4°Cto be used during nuclear isolation experiment. Human renal biopsies (5-12mg) werehomogenized using kits pestle on ice in 200ul of chilled lysis buffer supplemented with additional 0.2U/ulRNase inhibitor.The samples were incubated on ice from 10 min in additional 300ul of lysis buffer with0.2U/ulRNase inhibitor. The homogenate was transfer to a Nuclei Isolation Column in a 10%BSA precoated collection tube and centrifuged at 16,000rcf for 20sec at4°C. Column was then discarded and the supernatant was resuspended with gentle mixing 15xtimeswith pipet,then centrifuged at 500 x for 3min at 4°C. The pellet was resuspended and washed with 500ul of the Debris Removal Buffer with.2U/ul of RNase inhibitorand mixed by gently pipetting 15x times. The resuspension was then centrifuged at 700 rcf for 10min at 4°C and supernatant was discarded. The pellet was resuspended in 200ul of Wash Buffer and filtered through a 40-μm FLOWMI cell strainer (SP Bel-Art, 136800040) and counted using a hemacytometer. Samples with more than 1 million nuclei were then FACS sorted to clean up the sample using Sytox-7AAD Live dead staining. Samples with below 1 million nuclei after washing were filtered a second time with the 40-μm FLOWMI cell strainer. Gene libraries from isolated human renal single nuclei were constructed with droplet-based single-cell RNA sequencing using the Chromium Next Gen Single Cell 3' Reagent v3.1 Dual Index Kit. Single nuclei were load at 1,000 nuclei/ul for a targeted 10,000 nuclei per sample depending on starting biopsy weight.