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| Status |
Public on May 27, 2024 |
| Title |
Multiome rgc:ntr preablation wildtype control day 5 ATAC |
| Sample type |
SRA |
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| Source name |
Eye
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| Organism |
Danio rerio |
| Characteristics |
tissue: Eye
transgenic line: rgc:ntr
treatment: no mtz
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
scRNAseq: 40-60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain (10 eyes per 1 ml) (Worthington), and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin (Sigma-Aldrich) and 10 U/ml DNaseI (Roche). Cells were filtered through a 70-μm filter (Miltenyi Biotec), kept on ice until 10X genomics processing. scMultiomeseq: 40-60 eyes were dissected and flash-frozen in dry ice for ~15min before being transferred to a -80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression (GEX) protocol (CGOOO338). Briefly, frozen retinal tissues were lysed in ice-cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged, washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of ~3000-5000 nuclei/ml and kept on ice until 10x genomics processing.
Library preparation was then performed according to 10x genomics protocols.
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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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| Description |
TH182A
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| Data processing |
scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline (v4.3.0.1) and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs, and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly, the top 2,000 variable genes were identified and used to identify principal components (PCs) of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes (DEGs) were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster (minimum log2 foldchange cutoff of 0.25).
scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC-seq reads was performed using MACS2 in the ArchR package (v1.0.2). ATAC seq data was then processed using the pbmc scATAC-seq workflow with the Signac (v1.10.0) and Seurat (v4.3.0.1) packages for quality control, normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next, the ChromVar package (v1.18.0) was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells.
Assembly: GRCz11
Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes, features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files
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| Submission date |
May 23, 2024 |
| Last update date |
May 27, 2024 |
| Contact name |
Kevin Emmerich |
| E-mail(s) |
kemmeri2@jhmi.edu
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| Organization name |
Johns Hopkins University
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| Department |
Ophthalmology
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| Lab |
Jeff Mumm
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| Street address |
1800 Orleans St
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| City |
Baltimore |
| State/province |
Maryland |
| ZIP/Postal code |
21287 |
| Country |
USA |
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| Platform ID |
GPL24995 |
| Series (1) |
| GSE268179 |
Large-scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific |
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| Relations |
| BioSample |
SAMN41506332 |
| SRA |
SRX24663072 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8287444_TH182_atac_fragments.tsv.gz |
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(ftp)(http) |
TSV |
| GSM8287444_TH182_atac_fragments.tsv.gz.tbi.gz |
784.8 Kb |
(ftp)(http) |
TBI |
SRA Run Selector |
| Raw data are available in SRA |
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