Dataset DOI: 10.5061/dryad.tb2rbp0g3

Description of the data and file structure

This dataset is associated with Chiu et al. 2025, Glial betaPix is essential for blood vessel development in the zebrafish brain.

In this study, single-cell transcriptome libraries were obtained from embryonic zebrafish heads of wild-type siblings and betaPix CRISPR mutants at 1 dpf and 2 dpf. 300 ng/µL Cas9 mRNA and a mixture of number 1 to 4 betaPix gRNAs with 50 ng/µL each were co-injected into wildtype zebrafish embryos at one-cell stage. Injection with equivalent concentration of Cas9 mRNA with PBS served as siblings. After microinjection, embryos were collected and maintained at 28.5°C. At 24 or 48 hpf, zebrafish were dechorionated, paralyzed and transferred onto agarose plate. Heads were harvested with dissecting scissors in cold sterile 1X PBS with pooling 200 heads for each group. Heads were then dissociated in 900 µL Accutase cell detachment solution (Sigma-Aldrich, A6964) at 28.5°C for 3 hours and re-suspended by pipetting every 30 min. Once digestion was complete, 100 µL FBS was added to cell suspension and centrifuged at 4°C, 500 g for 3 min. The cells were gently re-suspended in cold 500 µL 2.5% FBS in IX PBS and filtered through a 40 µm strainer. PI and Hoechst33342 were stained for distinguishing living cells.

The single, living cells were sorted by Aria SORP (BD biosciences) into 1.5 ml tubes. The cell counts and vitality were verified by AOPI staining coupled with an Automated Cell Counter (Countstar BioTech). Around 10,000-12,000 cells were loaded for each group. Single cells were barcoded with Chromium Next GEM Single Cell 3 ‘Reagent Kits V3.1 kit (10x Genomics, 1000269) in 10× Chromium Controller (10× Genomics). After qualified by peak shapes, fragment size and tailing with Fragment Analyzer System kit (Agilent Technologies, DNF-915), single-cell transcriptome libraries were sequenced via Illumina High Throughput Sequencing PE150 (Novogene, Beijing).

Files and variables

The raw data files are named using a composite identifier that provides essential information about the sample type, developmental stage, library, and sequencing run. All files are raw single-cell RNA sequencing data in compressed FastQ format.

The file names follow the general structure: [Treatment]-[Developmental_Stage]-[Novogene_Accession]-[Sample_ID]_[Read_Pair].fq.gz

1 dpf samples are associated with Novogene Library Accession Numbers FKDL210231122-1a (Control) and FKDL210231123-1a (Knockout).

2 dpf samples are associated with Novogene Library Accession Numbers FKDL210125450-1a (Control) and FKDL210125451-1a (Knockout).

Example File Interpretation:

ko-2d-ko-2d_FKDL210125451-1a-AK3562_1.fq.gzDescription: Raw scRNA-seq data from a $betaPix$ crispant (ko) collected at 2 days post-fertilization (2d). It belongs to Novogene Library FKDL210125451-a, uses Index AK3562, and contains the first read (1) of the paired-end sequence.

Code/software

The sequencing data were analyzed using the CellRanger-6.1.1 (10x Genomics) and mapped to reference genome GRCz1l-GRCz11.103. Low-quality cells were excluded from subsequent analyses under the following conditions: when the number of expressed genes was less than 500, when there were abnormally high counts of UMIs or genes (outliers of a normal distribution), or when the mitochondrial content exceeded 9%. A total of 38,670 cells were qualified for subsequent analyses.

5’ adapter (5'-3'): AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACAC GACGCTCTTCCGATCT

3’ adapter (5'-3'): AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC（INDEX） ATCTCGTATGCCGTCTTCTGCTTG

ctrl-1d index (5'-3'): GCATCTCC; TGTAAGGT; CTGCGATG; AACGTCAA

ctrl-2d index (5'-3'): GTCTCTCG; AATCTCTC; CGGAGGGA; TCAGAAAT

ko-1d index (5'-3'): AGCTATCA; CATATAAC; TCAGGGTG; GTGCCCGT

ko-2d index (5'-3'): AAACCTCA; GCCTTGGT; CTGGACTC; TGTAGAAG