GSM5732393: transplanted hCS at day 224 of differentiation; Homo sapiens; RNA-Seq
Nuclei isolation was performed as described in (Matson et al., 2018) with some modifications. Briefly, t-hCS and hCS were disrupted using the detergent-mechanical cell lysis method with a 2 ml glass tissue grinder (Sigma-Aldrich/KIMBLE, D8938). Crude nuclei were then filtered using a 40 μm filter and centrifuged at 320 x g for 10 minutes at 4°C before performing a sucrose density gradient to separate them from cellular debris. After a centrifugation step (320 x g, 20 minutes at 4°C), samples were resuspended in 0.04% BSA/PBS supplemented with 0.2 U/μl RNAse inhibitor (Ambion 40U/μl, AM2682) and passed through a 40 μm flowmi filter. Dissociated nuclei were then resuspended in PBS containing 0.02% BSA and loaded onto a Chromium Single cell 3' chip (with an estimated recovery of 10,000 cells per channel). snRNA-seq libraries were prepared with the Chromium Single cell 3' GEM, Library & Gel Bead Kit v3 (10x Genomics).