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| Status |
Public on Jun 17, 2021 |
| Title |
EZH1 KO rep1 |
| Sample type |
SRA |
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| Source name |
kdrl:GFP+ cells 30hpf
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| Organism |
Danio rerio |
| Characteristics |
genotype: ezh1-/-
strain: Tg(kdrl:EGFP)
age: 30hpf
cell type: kdrl:GFP+ cells
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| Extracted molecule |
total RNA |
| Extraction protocol |
Pools of fluorescent zebrafish embryos/larvae were dissociated using Liberase TM (Sigma) in a 34C circulating water bath and resuspended in 1X PBS/1mM EDTA. kdrl:GFP+ cells were FACS sorted on a FACSAria II (BD) at the BCH Flow Cytometry Core into 1X PBS containing 2% fetal bovine serum.
Single-cell RNA-seq was performed on the 10X Genomics Chromium platform using the single cell expression 3’ v2 profiling chemistry. Following kdrl:GFP+ FACS, cells from each ezh1 genotype were loaded across two lanes in a 10X Genomics Single Cell 3’ Chip as independent technical replicates and libraries were constructed according to the manufacturer’s protocol. Cells were loaded into 10X lanes at cell concentrations to maintain an estimated doublet rate below 5%. The final libraries were assayed via an Agilent High Sensitivity dsDNA Bioanalyzer, normalized, pooled and shallow sequenced (Illumina MiniSeq), identifying ~11,000 high confidence cell barcodes in total across all libraries. The libraries were subsequently renormalized per the distribution of reads/library from the shallow sequencing run and deep sequenced on a NovaSeq S4 (Illumina) to a depth of ~100,000 reads per cell with cycle configuration 151-8-8-151.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
A custom Danio rerio reference genome containing a GFP transcript was generated using Ensembl Release 101 (ftp://ftp.ensembl.org/pub/release-101/gtf/danio_rerio/) with the mkref command from cellranger v2.1 (10X Genomics)
The raw sequencing files were aligned to the custom reference processed with the Cellranger count pipeline.
Cellranger count matrices for each library were read into R (v3.5) and analyzed with Seurat v3.2. High-quality cells were retained for subsequent analyses based on technical metrics: <5% mitochondrial reads, >1000 UMI counts, and >500 genes detected. A Seurat object of all unfiltered cells across all libraries is provided as a supplementary file.
Genome_build: GRCz11-101
Supplementary_files_format_and_content: Cells by genes count matrices produced from the cellranger pipeline
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| Submission date |
May 06, 2021 |
| Last update date |
Jun 19, 2021 |
| Contact name |
Mohamad Ali Najia |
| E-mail(s) |
[email protected]
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| Organization name |
Boston Children's Hospital
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| Department |
Hematology/Oncology
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| Lab |
George Daley
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| Street address |
1 Blackfan Circle
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL24995 |
| Series (1) |
| GSE173972 |
Differential Ezh1/2 regulation of hemogenic fate and hematopoietic stem and progenitor cell formation from arterial endothelium |
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| Relations |
| BioSample |
SAMN19033027 |
| SRA |
SRX10807781 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5283743_EZH1_KO_1_barcodes.tsv.gz |
6.9 Kb |
(ftp)(http) |
TSV |
| GSM5283743_EZH1_KO_1_genes.tsv.gz |
202.1 Kb |
(ftp)(http) |
TSV |
| GSM5283743_EZH1_KO_1_matrix.mtx.gz |
8.4 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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