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| Status |
Public on Sep 27, 2023 |
| Title |
HB13hpf_rep2_ATAC |
| Sample type |
SRA |
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| Source name |
dissected hindbrain region
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| Organism |
Danio rerio |
| Characteristics |
tissue: dissected hindbrain region
genotype: wildtype(TU line obtained from ZIRC stockcenter)
treatment: untreated
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| Treatment protocol |
10hpf and 16hpf samples were left untreated. One 13hpf sample was left untreated, the other 13hpf sample was treated with 5uM DEAB (4-Diethylaminobenzaldehyde) from 4hpf until 13hpf.
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| Growth protocol |
Zebrafish embryos were generated by natural spawnings and grown under standard conditions
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Tissue was collected in 1XPBS, dissociated by repeated pipetting through a p1000 tip, and centrifuged at 4℃ at 400G for 5 minutes followed by resuspension of the pellet in 500 μl of protease (10 mg/ml BI protease (Sigma, P5380), 125 U/ml DNase, 2.5 mM EDTA in PBS) for 15minutes on ice. Samples were centrifuged at 4℃ at 400G for 5 minutes, resuspended in 1ml HBSS + FBS (2%), filtered through a 20 μm cell strainer (pluriSelect, KL-071912), recentrifuged at 4℃ at 400G for 5 minutes, resuspended in 500 μl of HBSS + FBS (2%) and again filtered through a 20 μm cell strainer. Following centrifugation at 4℃ at 400G for 5 minutes, the cells were resuspended in 200 μl PBS. For nuclei isolation, we followed the 10X Genomics recommended protocol with minor modifications. The dissociated cells were centrifuged at 900G at 4℃ for 5 minutes, resuspended in 100 μl of 0.1X lysis buffer (1 mM Tris-HCl pH7.4, 1 mM NaCl, 0.3 mM MgCl2, 0.1% BSA, 0.01 % Tween-20, 0.01 % NP40, 0.001 % Digitonin (Invitrogen, BN2006), 0.1 mM DTT, 0.1 U/μl RNase inhibitor, in nuclease-free water) and incubated on ice for 5 min. Samples were then washed three times by centrifugation at 4℃ at 900G for 10 min and resuspension in 1 ml of chilled wash buffer (10 mM Tris-HCl pH7.4, 10 mM NaCl, 3 mM MgCl2, 1% BSA, 0.1 % Tween-20, 1 mM DTT, 1 U/μl RNase inhibitor in nuclease-free water). Finally, nuclei were counted and resuspended in 1X nuclei buffer (provided in the 10x Genomics Single Cell Multiome ATAC Kit A; 1 mM DTT, 1 U/μl RNase inhibitor in nuclease free water) at a final concentration of approximately 2,000-3,000 nuclei/μl for sequencing on the 10X scMultiome platform.
10x Genomics Single Cell Multiome ATAC Kit A was used with the standard protocol
Targets of 100,000 reads each GEX and ATAC per cell and >= 2,000 cells per sample.
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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 |
10x Genomics multiomic, ATAC-seq
hindbrain 13hpf rep2
HB13hpf_rep2.clustered.RDS
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| Data processing |
Generated cell feature counts for each single cell RNAseq library using cellranger arc count --reference=GRCz11
single cell RNAseq feature and ATACseq feature data generated using Signac/Seurat Read10X_h5 and SeuratFromMatrixh5 functions using GRCz11.v99.EnsDb annotation
filtered Seurat object to select cells with nCount_ATAC>1000 & nCount_RNA >500 & percent.mitchondrial <5% & nucleosome_signal<2 & TSS.enrichment>1
generated ATAC peaks with Signac CallPeaks function using macs2 and used in CreateChromatinAssay with name "peaks"
processed filtered Seurat object using Seurat SCTransform, RunPCA, FindTopFeatures, RunTFIDF, RunSVD, FindMultiModalNeighbors(reduction.list=list("pca","lsi"),dims.list=list(1:50,2:50),FindClusters(various resolutions) and RunUMAP(reduction="pca",dims=1:50)
Assembly: GRCz11
Supplementary files format and content: h5 files are Hierarchical Data Formats files of cellranger_arc count matrix output
Supplementary files format and content: tsv.gz files are gzipped tab delimited bed files of atac fragments
Supplementary files format and content: RDS files are R file containing SeuratObject
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| Submission date |
Sep 26, 2023 |
| Last update date |
Sep 27, 2023 |
| Contact name |
rebecca L orourke |
| E-mail(s) |
Rebecca.ORourke@cuanschutz.edu
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| Organization name |
University of Colorado Anschutz SOM
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| Street address |
12800 E 19th Ave
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| City |
Aurora |
| State/province |
CO |
| ZIP/Postal code |
80045 |
| Country |
USA |
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| Platform ID |
GPL24995 |
| Series (1) |
| GSE223535 |
scMultiome analysis identifies embryonic hindbrain progenitors with mixed rhombomere identities |
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| Relations |
| BioSample |
SAMN37547331 |
| SRA |
SRX21896000 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7806730_HB13hpf_rep2_atac_fragments.tsv.gz |
201.2 Mb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
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