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| Status |
Public on Jun 08, 2023 |
| Title |
Control, periphery, retina (scATAC-seq) |
| Sample type |
SRA |
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| Source name |
neural retina
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| Organism |
Homo sapiens |
| Characteristics |
individual: control
tissue: neural retina
region: peripheral
genotype: m.3243A
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Neural retina was dissected away from the underlying retinal pigment epithelium and choroid prior to tissue dissociation. Neural retina samples were dissociated with 20 units/mL of papain (Worthington Biochemical Corporation) with 0.0005% DNase I (Worthington Biochemical Corporation) on a shaker at 37˚C for 75 minutes. RPE/choroid samples were dissociated mechanically using razor blades into one-millimeter pieces then dissociated enzymatically using collagenase on a shaker at 37˚C for 60 minutes. Following dissociation, cells were cryopreserved in DMSO-based Recovery Cell Cryopreservation Media (Gibco). After overnight cooling at -80˚C, samples were transferred to liquid nitrogen (vapor phase) for long-term storage prior to encapsulation and library preparation.
Cells were prepared for mt-scATAC sequencing using the protocol described previously by Lareau, Ludwig et al. Briefly, cells were thawed as above and filtered through a 70µm cell strainer. Cells were then washed twice with dPBS-/- (Gibco). Cells were fixed with 1% formaldehyde (Sigma) in PBS-/- for 10 minutes at room temperature. Fixation was quenched with the addition of glycine (Research Products International) to a final concentration of 0.125M for 5 minutes at room temperature. Following fixation, cells were washed twice with ice-cold PBS-/-. Following the second wash, cells were resuspended in 100µL ice-cold Lysis Buffer (10 mM Tris-HCl pH 7.4 (Millipore-Sigma), 10 mM NaCl (Ambion/Invitrogen), 3 mM MgCl2 (Ambion/Invitrogen), 0.1% Nonidet-P40 substitute (Research Products International), 1% Fraction V BSA (Research Products International)) and incubated on ice for 2.5 minutes. Following incubation, 1mL of ice-cold Wash Buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1% BSA) was added, and cells were pelleted at 500 x g for 5 minutes at 4˚C. Cells were resuspended in 10µL diluted Nuclei Buffer (10X Genomics) and counted using Trypan Blue (Gibco) with the Countess automated cell counter (Invitrogen). Based on these counts, cell suspensions were diluted in diluted Nuclei Buffer to a final target concentration of 6,000 cells per µL. Cells were then processed following the 10X Chromium Next GEM Single Cell ATAC Reagent Kit v1.1 User Guide (Rev. D) without modification. Final libraries were quantified using the Qubit dsDNA HS Assay Kit (Life Technologies) and diluted to 3ng/µL. Library quality was checked using the Bioanalyzer instrument (Agilent), wherein periodicity of fragment length was observed, consistent with high quality ATAC-seq library construction.
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| Library strategy |
ATAC-seq |
| Library source |
genomic single cell |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
FASTQ files were generated from base calls with the bcl2fastq software (Illumina).
mtscATAC-seq reads were mapped to a modified GRCh38 reference genome that was generated by hard-masking nuclear regions that share homology with mitochondrial sequences, as described previously (39). Mapping and quantification were carried out by Cell Ranger ATAC (v2.0.0, 10X Genomics) using the ‘count’ function.
ATAC peaks for each sample were filtered using Signac (v1.4.0). Peaks with widths less than 20 or greater than 10000 were removed. Objects containing fragments for each sample were generated
Cells were filtered with the Signac (v1.4.0) subset function. Cells with fewer than 1000 peak region framents or greater than 75000 peak region framents were removed. Cells with less than 10x average mtDNA depth or greather than 1000x mtDNA depth were also removed. Mitochondrial DNA sequencing depth (mtDNA_depth) was calculated using mgatk.
Term frequency inverse document frequency (TF-IDF) normalization and partial singular value decomposition were performed on each library using RunTFIDF() and RunSVD() (Signac) with default settings. Gene activities were computed using GeneActivity().
Integration anchors between datasets were identified using reciprocal LSI projection using FindIntegrationAnchors() in Signac. Dimensions 2-30 from the CCA were used. LSI embeddings were integrated using IntegrateEmbeddings based on these anchors and using dimensions 1-30. A UMAP was generated using the integrated embeddings using RunUMAP(integrated_object, reduction = "integrated_lsi", dims = 2:30).
Cell identity was transferred from corresponding scRNAseq data of the same samples using TransferData() (Signac). Transfer anchors between RNA and ATAC data were identified using FindTransferAnchors with the following parameters: reduction = 'cca', dims = 1:40.
Assembly: hg38
Supplementary files format and content: Processed gene activity data matrix files are provided in comma-delimited format for count (*_count.csv) and processed/normalized (*_normalized.csv) activity values. For processed/normalized files, log-normalized activity values (from GetAssayData(object = seurat_object)) were appended to relevant metadata (barcode, cluster label, and donor number from the manuscript). Each row represents a unique cell, and columns correspond to metadata and log normalized gene activity values. For count files, raw gene activity level were obtained (from GetAssayData(object = seurat_object, slot = “counts”)) for each cell type in each cluster and appended to relevant metadata (barcode, cluster label, and donor number from the manuscript).
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| Submission date |
May 11, 2022 |
| Last update date |
Jun 08, 2023 |
| 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 (2) |
| GSE202746 |
Chromatin accessibility and mitochondrial genotype from single cells of the retina and choroid in human MELAS (m.3243A>G) and control samples [scATAC-seq] |
| GSE202747 |
Non-random distribution of mitochondrial m.3243A>G heteroplasmy in human retina and its impact on cellular phenotype |
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| Relations |
| BioSample |
SAMN28192771 |
| SRA |
SRX15234901 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6132187_20_034_peripheral_retina_counts.csv.gz |
26.0 Mb |
(ftp)(http) |
CSV |
| GSM6132187_20_034_peripheral_retina_normalized.csv.gz |
35.3 Mb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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