GSM8181645: retinal organoid, NR2E3-null, D80, scRNAseq; Homo sapiens; RNA-Seq
Organoid dissociation for single-cell RNAseq: For Day 40-260 scRNAseq samples, approximately ten organoids displaying morphology of successful retinal differentiation were selected for each of the three lines. Organoids were allowed to settle by gravity in a 1.5mL tube and culture medium was removed. Organoids were dissociated in a solution of 20U/mL papain (Worthington) and 120U/mL DNase (Worthington) in Earle's balanced salt solution (EBSS) (Worthington). Samples were incubated in 300µL of above papain solution at 37˚C with continuous shaking (500rpm) until all organoids were completely dissociated (approximately 1 hour). Samples were triturated with a pipette every 15 minutes. Following dissociation, cells were pelleted at 500 x g for 5 minutes and resuspended in 8µg/mL recombinant albumin (New England Biolabs) in dPBS-/-. Cells were passed through a 70µm filter to encapsulation with the Chromium Controller instrument (10X Genomics). Approximately 8,000 cells were targeted for encapsulation per sample.   Nucleus isolation for single-nucleus multimodal sequencing: Nuclei were isolated from differentiation day 160 and 260 organoids from an independent batch of differentiation (i.e., completely different round initiated on a separate day) than single-cell gene expression samples. Nuclei were isolated following a protocol based on 10X Genomics' demonstrated protocol CG000366. Briefly, approximately ten organoids were selected as described above. Medium was aspirated and 500µL of chilled 0.1X Lysis Buffer (Tris-HCl, NaCl, MgCl2, Tween-20, NP40, Digitonin, BSA, DTT, RNase Inhibitor) was added to each sample. Samples were homogenized with 15 strokes of a sterile pestle in a 1.5mL tube on ice. Samples were then incubated on ice for 5 minutes. Following incubation, samples were mixed with a P1000 pipette 10 times on ice and incubated for 8.5 minutes on ice. 500µL of Wash Buffer (Tris-HCl, NaCl, MgCl2, Tween-20, BSA, DTT, RNase Inhibitor) was added and lysed cells were mixed 5 times with pipetting. Cells were pelleted at 500 x g for 5 minutes in a refrigerated (4˚C) centrifuge and resuspended in 500µL chilled wash buffer. This process was repeated for three total washes. Nuclei were resuspended in Diluted Nuclei Buffer (Nuclei Buffer (10X Genomics), DTT, RNase Inhibitor) and passed through a 70µm strainer. Nuclei were counted on a hemocytometer with DAPI to visualize intact nuclei. Approximately 9,000 nuclei were targeted for encapsulation per sample using the Chromium X instrument (10X Genomics). Single-cell gene expression library preparation and sequencing: Single cells were partitioned and barcoded with the Chromium Controller instrument (10X Genomics) and Single Cell 3' Reagent (v3.1 chemistry) kit (10X Genomics) according to the manufacturer's specifications with no modification (Rev C). Final libraries were quantified using the Qubit dsDNA HS Assay Kit (Life Technologies) and diluted to 3ng/µL in buffer EB (Qiagen). Library quality was confirmed using the Bioanalyzer High Sensitivity DNA Assay (Agilent) prior to sequencing.  Single-nucleus multimodal library preparation and sequencing: Nuclei were processed following the 10X Genomics Chromium Next GEM Single Cell Multiome ATAC + Gene Expression (Rev. E) without modification. Final libraries were quantified using the Qubit dsDNA HS Assay Kit and diluted to 3ng/µL. Library quality was checked using the Bioanalyzer or Tapestation (Agilent).