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| Status |
Public on Jun 06, 2023 |
| Title |
SP_36hpf |
| Sample type |
SRA |
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| Source name |
Zebrafish trunk region
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| Organism |
Danio rerio |
| Characteristics |
tissue: Zebrafish trunk region 36hpf
cells: single spi2: Gal4;UAS:GFP+ kdrl:mCherry+ HECs and spi2: Gal4;UAS:GFP+ kdrl:mCherry- hematopoietic cells
Stage: 36 hours post fertilization
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| Extracted molecule |
total RNA |
| Extraction protocol |
For 10x Genomics-based scRNA-seq and scATAC-seq in zebrafish, 40,000 mCherry+ GFP- cells, 40,000 mCherry+ GFP+ cells and 30,000 mCherry- GFP+ cells were sorted from Tg (kdrl:mCherry/runx1:enGFP) at 36 hpf. For STRT-seq in zebrafish, single spi2: Gal4;UAS:GFP+ kdrl:mCherry+ HECs and spi2: Gal4;UAS:GFP+ kdrl:mCherry- hematopoietic cells were sorted from the trunk region of Tg (spi2: Gal4;UAS:GFP/ kdrl:mCherry) at 36 hpf. For scRNA-seq of spi2 morphants at 36 hpf in zebrafish, ECs (kdrl+runx1-), HECs (kdrl+runx1+) and hematopoietic cells (kdrl-runx1+) were sorted. For bulk CUT&TAG in zebrafish, fli1a-flag-spi2-EGFP+ cells were sorted from the trunk region of Tg (fli1a-flag-spi2-GFP) embryos at 36 hpf.
For 10x Genomics-based scRNA-seq and scATAC-seq in zebrafish, we loaded ~20,000 cells for further 10x Genomics-based scRNA-seq and ~90,000 cells for further 10x Genomics-based scATAC-seq. For scRNA-seq, libraries were prepared using Single Cell 3’ Library and Gel Bead Kit V3.1. Sequencing was performed on an Illumina Novaseq6000 platform to generate 150 bp paired-end reads. For ATAC-seq, nuclei were isolated and washed according to the methods supplied by 10x Genomics. Libraries were prepared using the Chromium Chip E Single Cell Kit and Chromium Single Cell ATAC Library & Gel Bead Kit, and further sequenced on an Illumina Novaseq6000 platform to generate 50 bp paired-end reads. For 10x Genomics-based scRNA-seq in mice, libraries were prepared using Single Cell 3’ Library and Gel Bead Kit V3.1. Sequencing was performed on an Illumina Novaseq6000 platform to generate 150 bp paired-end reads. For STRT-seq in zebrafish, the end-repair and dA-tailing of the DNA fragments and ligation of the adaptors to the DNA fragments were performed according to the KAPA Hyper Prep Kits with PCR Library Amplification/Illumina series. After the adaptor ligation step, the final PCR was performed. The libraries were sequenced on an Illumina Novaseq6000 platform to generate 150 bp paired-end reads. For bulk CUT&TAG, libraries were prepared according to Hyperactive In-Situ ChIP Library Prep Kit for Illumina and sequenced on an Illumina NovaSeq6000 platform to generate 150 bp paired-end reads.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
For scRNA-seq and scATAC-seq based on 10x Genomics，raw data files were processed by Cell Ranger software suite with default mapping parameters, using the GRCz11 reference genome.
For STRT-seq, raw reads were first de-multiplexed by barcode sequences in reads 2 to yield separate read files for individual cells, then the transcripts sequences of each cell in reads 1 were separated based on corresponding reads 2. Simultaneously, UMI sequences in reads 2 were integrated into reads 1. The template switching oligo (TSO) sequence, polyA sequence and the low-quality reads (N > 10%) in reads 1 were subsequently removed by Python scripts and Trimmomatic (version 0.36). Next, the clean reads were aligned to the zebrafish genome (GRCz11 from Ensembl) using HISAT2 (version 2.1.0) with known gene annotation. Only protein-coding genes were retained and the abundance of each gene were estimated by counting the reads that duplicated UMIs have been excluded.
For cut&tag, reads were aligned to GRCz11 by Bowtie2. Only uniquely mapped reads with mapping quality score ≥ 30 were kept using Samtools software. After merging replicates, MACS2 was used for the peak calling.
Assembly: GRCz11
Library strategy: STRT-seq
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| Submission date |
Mar 06, 2023 |
| Last update date |
Jun 06, 2023 |
| Contact name |
Zhixin Kang |
| E-mail(s) |
kzhixcas@gmail.com
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| Phone |
+86-01064807562
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| Organization name |
INSTITUTE OF ZOOLOGY, CHINESE ACADEMY OF SCIENCES
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| Lab |
Feng Liu
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| Street address |
1 Beichen West Road, Chaoyang District
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| City |
Beijing |
| ZIP/Postal code |
100101 |
| Country |
China |
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| Platform ID |
GPL24995 |
| Series (2) |
| GSE186425 |
Activation of lineage competence in hemogenic endothelium precedes the formation of hematopoietic stem cell heterogeneity [Zebrafish.STRT-seq] |
| GSE186427 |
Activation of lineage competence in hemogenic endothelium precedes the formation of hematopoietic stem cell heterogeneity |
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| Relations |
| BioSample |
SAMN33603801 |
| SRA |
SRX19578257 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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