GSM7383221: Renal organoids, d10, multi-ome-seq, batch 3; Homo sapiens; OTHER
Single cell preparations were generated as above, then further processed for single nucleus isolation by centrifugation at 300g for 4 min at 12oC and resuspension in 100µL freshly prepared ice-cold lysis buffer (10mM Tris-HCL, 10mM NaCl, 3mM MgCl2, 0.1% Tween-20, 1 % BSA, 1mM DTT, 1 U/µL Protector RNase Inhibitor (Sigma, 3335402001), 0.1% Nonidet P40 Substitute (Sigma, 74385) and 0.01% Digitonin in nuclease-free H2O) followed by incubation on ice for 5 min. Lysis was halted by addition of 900µL chilled wash buffer (10mM Tris-HCL, 10mM NaCl, 3mM MgCl2, 0.1% Tween-20, 1 % BSA, 1mM DTT, 1 U/µL Protector RNase Inhibitor in H2O), followed by 3 rounds of centrifugation at 500g for 5 min at 4oC and resuspension in chilled wash buffer, with the final resuspension in 50µL chilled 1x Nuclei Buffer (10X Genomics, 2000153) with 1mM DTT and 1 U/µL Protector RNase Inhibitor in H2O, according to the 10x Genomics demonstrated protocol for Nuclei Isolation for Single Cell Multiome (ATAC + GEX) sequencing (CG000365, RevA). After resuspension in 1x Nuclei Buffer, nuclei were stained with Trypan Blue and counted using a Countess II (Life Technologies) to confirm complete lysis and to achieve a targeted nuclei recovery of 1,600 nuclei per sample for batch 2, and 5,000 nuclei per sample for batch 3. Nuclei were processed for library preparation using a 10X Chromium scMultiome kit (10X Genomics) at the Cancer Research UK Genomics Facility.