GSM5016323: day 63 kidney organoid snRNA-seq; Homo sapiens; RNA-Seq
Nuclei were isolated with Nuclei EZ Lysis buffer (NUC-101; Sigma-Aldrich) supplemented with protease inhibitor (5892791001; Roche) and RNase inhibitor (N2615; Promega and AM2696; Life Technologies). Organoids were homogenized using a Dounce homogenizer (885302–0002; Kimble Chase) in 2 ml of ice-cold Nuclei EZ Lysis buffer, and they were incubated on ice for 5 minutes with an additional 2 ml of lysis buffer. The homogenate was filtered through a 30 mm cell strainer (43– 50030–03; pluriSelect) and then centrifuged at 500g for 5 minutes at 4°C. The pellet was resuspended and washed with 4 ml of the buffer, and then, it was incubated on ice for 5 minutes. After another centrifugation, the pellet was resuspended in Nuclei Suspension Buffer (13 PBS, 0.04 % BSA, and 0.1% RNase inhibitor), filtered through a 5 mm cell strainer (43–50005–03; pluriSelect), and counted. RNA from single nucleus was encapsulated, barcoded and reversed transcribed on a 10× Chromium Single Cell Platform (10× Genomics).