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| Status |
Public on Apr 01, 2024 |
| Title |
retinal organoid, NR2E3-null, D260, scRNAseq |
| Sample type |
SRA |
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| Source name |
NR2E3-null
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| Organism |
Homo sapiens |
| Characteristics |
sample type: retinal organoid
cell line: NR2E3-null
differentiation day: D260
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| Growth protocol |
Retinal differentiation was performed as described previously with minor modifications (Bohrer 2023). Briefly, iPSCs were cultured on laminin 521 coated plates in E8 medium. Embryoid bodies (EBs) were lifted with ReLeSR (STEMCELL Technologies, Cambridge, MA) and transitioned from E8 to neural induction medium (NIM- DMEM/F12 (1:1), 1% N2 supplement, 1% non-essential amino acids, 1% Glutamax (Thermo Fisher Scientific), 2 µg/mL heparin (Sigma-Aldrich, St. Louis MO) and Primocin (InvivoGen, San Diego, CA)) over a four-day period. On day 6, NIM was supplemented with 1.5 nM BMP4 (R&D Systems, Minneapolis, MN). On day 7, EBs were adhered to Matrigel coated plates (Corning). BMP4 was gradually transitioned out of the NIM over seven days. On day 16, the media was changed to retinal differentiation medium (RDM - DMEM/F12 (3:1), 2% B27 supplement (Thermo Fisher Scientific), 1% non-essential amino acids, 1% Glutamax and 0.2% Primocin). On day 25-30 the entire EB outgrowth was mechanically lifted using a cell scraper and transferred to ultra-low attachment flasks in 3D-RDM (RDM plus 10% Fetal bovine serum (FBS)); Thermo Fisher Scientific), 100 µM taurine (Sigma-Aldrich), 1:1000 chemically defined lipid concentrate (Thermo Fisher Scientific), and 1 µM all-trans retinoic acid (until day 100; Sigma-Aldrich). The cells were fed three times per week with 3D-RDM until harvest.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
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).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Single-cell gene expression data integration and processing: scRNA libraries were pooled and sequenced using the NovaSeq 6000 instrument (Illumina) generating 100-bp paired end reads. FASTQ files were generated from base calls with the bcl2fastq software (Illumina). For the ESCS Patient 2 experiment, reads were mapped to the pre-built GRCh38 reference (refdata-gex-GRCh38-2020-A) with Cell Ranger v7.0.0 (10X Genomics) using the ‘count’ function with the following parameters: --expect-cells=8000 --localcores=56. Only cells passing the default Cell Ranger call were analyzed further. For the AAV experiment, custom reference genomes were built to allow mapping of EGFP and associated 3’UTR regions originating from AAV cargo. For AAV- NR2E3-T2A-EGFP transduced and untransduced samples, reads were mapped to a reference genome containing T2A-EGFP-BGHpolyA sequence. For AAV-EGFP transduced samples, reads were mapped to a reference genome containing EGFP-WPRE-BGHpolyA sequence. Data from all samples was integrated using CCA. The top 2000 variable features as determined by vst method were used. For AAV-transduced samples, the transgene feature was not included as a variable feature. Only cells with between 1,000 and 7,000 unique genes (features) were included in the analysis. Only cells with < 10% of reads mapping to mtDNA-encoded genes and < 20% of reading mapping to ribosomal genes were included. Counts data were normalized using the NormalizeData function (Seurat) with the following parameters: normalization.method = "LogNormalize", scale.factor = 10000. 2000 variable features were identified with the FindVariableFeatures function using the vst selection method. Integration anchors were identified using the FindIntegrationAnchors function using 25 dimensions. An assay “Integrated” was generated for the 2000 variable features using the IntegrateData function using 25 dimensions. The integrated data were then used in principal component analysis (PCA).
Single-nucleus multimodal data integration and processing: Single-nucleus multimodal libraries were sequenced using the NovaSeq 6000 instrument (Illumina). Sequencing was performed by the Genomics Division of the Iowa Institute of Human Genetics. FASTQ files were generated from base calls with the bcl2fastq software (Illumina). Reads were mapped to the pre-built GRCh38 reference (GRCh38-2020-A-2.0.0, 10X Genomics) using Cell Ranger ARC (v.2.0.0, 10X Genomics) with default parameters. Resulting cell-by-peak and cell-by-gene matrices (ATAC and Gene Expression assays respectively) from the four samples were integrated separately using latent semantic indexing (ATAC) and canonical correlation analysis (gene expression) respectively. For CCA, only cells with between 500 and 4,000 unique genes (features) were included in the analysis. Only cells with < 10% of reads mapping to mtDNA-encoded genes and < 8% of reading mapping to ribosomal genes were included. For LSI, only cells with between 2,000 and 15,000 peak region fragments and less than 1,000 mtDNA depth were included in analysis. Cells passing both ATAC and Gene Expression QC cutoffs were retained and used to generate a UMAP following Weighted Nearest Neighbor (WNN) analysis. MultiModal Neighbors were identified using dimensions 1:30 from the PCA resulting from CCA (GEX) and 2:50 from the LSI reduction (ATAC). Clusters were identified using FindClusters (Seurat) based on the "wsnn" graph using the SLM algorithm with a resolution of 0.5. Clusters were annotted for cell type manually. ATAC peaks to be used downstream (i.e. motif enrichment analysis) were called using MACS2 (Feng et al., 2012; Zhang et al., 2008) with the CallPeaks function in Signac.
Dimensionality reduction with UMAP and cell type annotation: 30 principal components were identified out of the integrated dataset described above using the RunPCA function (Seurat) (Stuart et al., 2019). Uniform manifold approximation and projection (UMAP) was performed using the RunUMAP function using 25 principal components. Cells were annotated with the using the D40-D160 organoid dataset from this publication (Mullin, Bohrer et al., 2024) as a reference.
Assembly: GRCh38
Supplementary files format and content: For scRNAseq data, raw and log normalzied counts are provided for each library (*_counts.csv and *_normalized.csv, respectively). Counts were accessed from the filtered Seurat object via seurat_obj@assays$RNA@counts. Normalized counts were accessed from the filtered Seurat object via seurat_obj@assays$RNA@data. Count matrices are appended with the following metadata: "barcode", 10X cell barcode; "donor", iPSC line of origin; "timepoint", differentiation day of sample collection; "library", unique library identifier; "celltype", initial cell type annotation; "umap_dim_1"/"umap_dim_2", UMAP coordinates.
Supplementary files format and content: For multimodal sequencing data, raw and log normalzied gene expression (GEX) counts and raw peak counts (from MACS2 calls) are provided for each library (*_gex_counts.csv and *_gex_normalized.csv, and *_peaks_counts.csv respectively). Raw gene expression counts were accessed from the filtered Seurat object via seurat_obj@assays$RNA@counts. Normalized gene expression counts were accessed from the filtered Seurat object via seurat_obj@assays$RNA@data. Raw peak counts were accessed from the filtered Seurat object via seurat_obj@assays$macs2_peaks@counts. Count matrices are appended with the following metadata: "barcode", 10X cell barcode; "donor", iPSC line of origin; "timepoint", differentiation day of sample collection; "library", unique library identifier; "celltype", initial cell type annotation; "umap_dim_1"/"umap_dim_2", UMAP coordinates.
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| Submission date |
Apr 01, 2024 |
| Last update date |
Apr 01, 2024 |
| Contact name |
Nathaniel Kevin Mullin |
| Organization name |
University of Iowa
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| Street address |
375 Newton Road
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| City |
Iowa City |
| State/province |
IA |
| ZIP/Postal code |
52246 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE236197 |
NR2E3 loss disrupts photoreceptor cell maturation and fate in human organoid models of retinal development |
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| Relations |
| BioSample |
SAMN40709436 |
| SRA |
SRX24117689 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8181654_nr2e3_null_D260_counts.csv.gz |
26.9 Mb |
(ftp)(http) |
CSV |
| GSM8181654_nr2e3_null_D260_normalized.csv.gz |
34.2 Mb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
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