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| Status |
Public on Jun 07, 2023 |
| Title |
Spinal cord, saig, rep 1 |
| Sample type |
SRA |
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| Source name |
spine
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| Organism |
Danio rerio |
| Characteristics |
tissue: spine
cell type: mixed spinal cells
genotype: Tg(SAIG213A;EGFP)
developmental stage: 4 dpf
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| Extracted molecule |
total RNA |
| Extraction protocol |
About 150 4-dpf larva were euthanized in 0.02% tricaine, and individually decapitated behind the hindbrain. They were incubated with 20 mg/ml collagenase in bath solution (134 mM NaCl, 2.9 mM KCl, 1.2 mM MgCl2, 2.1 mM CaCl2, and 10 mM Na-HEPES, pH 7.8, ∼290 mOsm) at 28 °C for 1.5 hr, with intermittent trituration using a p200 pipette aid at 0, 0.5 hr and1 hr of the incubation. For the purpose of isolating spinal cords from remaining tissue, the final trituration was done using fire-polished Pasteur pipettes with decreased opening sizes (300, 200, 100 μm respectively). Intact spinal cords were transferred to L15 media and washed 3 times with fresh media. The spinal cords were incubated with 0.25% trypsin solution (in 1xPBS containing 1 mM EDTA) at 28 °C for 25 min. The digestion was terminated by adding 500 μl stop solution (L15 with 1% fetal bovine serum). The tissue was collected by spinning at 400 g for 3 min at 4°C, washed once with L15 and resuspended in 200 μl of L15 media. Spinal cord cells were dissociated by triturating the digested tissue with fire-polished Pasteur pipettes with 80-100 μm opening. The solution was filtered through a 35μm strainer into a siliconized collection tube. The suspension was examined on a microscope for cell count, Trypan blue staining based - viability test and proportion of dispersed single cells. Samples with a viability above 70% were used for sequencing. For FACS sorted samples single cell suspension was prepared as above from Tg(mnx1:GFP) fish and were FAC sorted for EGFP+ cells using a 100 μm nozzle on a BD inFlux cell sorter (Flow Cytometry Shared Resource, OHSU)
Single cell capture, cDNA synthesis and library preparation were performed by the Massive Parallel Sequencing Shared Resource at OHSU using the 10x Genomics Chromium v3.0 reagent kit. Single cell suspension for the two full spinal cord replicates targeted 10,000-15,000 cells, for the two FACS sorted samples 4,000-5,000 cells were targeted.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Cells dissociated from ~150 zebrafish spines
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| Data processing |
Barcode processing and gene counting were preformed using Cell ranger software v6.1.1 (10X Genomics). Reads were aligned to a reference genome made using cellrangers mkref command using the GRCz11.fa and the zebrafish_custom_ref.GTF provided as a supplimental file. The zebrafish_custom_ref.GTF is a lightly modified version of the Lawson v 1.4.3 annotation (Lawson et al, 2020, eLife) in which GFP has been added and a number of naming mistakes have been corrected.
Normalization, integration, and quality control were preformed using Seurat. Clustering of the data was preformed using the Lieden algorithm implemented in Seurat with varying parameters depending on the sample. Clusters with markers indicating contamination (hindbrain and muscle) were removed as were clusters where over 90% of cells came from only one experimental run.
Neurons and glia were isolated based on the expression of markers within clusters, snap25a and elavl4 for neurons and slc1a2b, gfap, myrf and sox10 for glia. Neurons were then sub-clustered into 33 clusters and identities were assigned to 21 of them based on both established markers in the literature and a number of novel markers we were able to validate.
Motor neurons were isolated in the dataset based on the expression of motor neuron markers mnx1, mnx2b, isl1, and cholinergic markers slc18a3a ,and chata within clusters. Motor neurons isolated this way were then integrated together using Seurat.
Assembly: GRCz11
Supplementary files format and content: Tab seperated value files, and matrix files providing the processed count matrices for each sample
Supplementary files format and content: RDS file providing the Seurat data object for the integrated spinal samples
Supplementary files format and content: RDS file providing the Seurat data object for the annotated set of clustered neurons from the integrated spinal samples
Supplementary files format and content: RDS file providing the Seurat data object for the integrated set of isolated motor neurons from all samples
Supplementary files format and content: GTF format record of the reference genome used in the creation of this data set
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| Submission date |
May 18, 2023 |
| Last update date |
Jun 07, 2023 |
| Contact name |
Paul Brehm |
| E-mail(s) |
brehmp@ohsu.edu
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| Organization name |
Oregon Health and Science University
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| Department |
Vollum
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| Lab |
Brehm
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| Street address |
3232 SW Research Drive
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| City |
Portland |
| State/province |
OR |
| ZIP/Postal code |
97239 |
| Country |
USA |
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| Platform ID |
GPL20828 |
| Series (1) |
| GSE232801 |
sc-RNAseq of the day 4 zebrafish spinal cord |
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| Relations |
| BioSample |
SAMN35158391 |
| SRA |
SRX20421318 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7383490_barcodes_saig_210928.tsv.gz |
113.0 Kb |
(ftp)(http) |
TSV |
| GSM7383490_features_saig_210928.tsv.gz |
208.5 Kb |
(ftp)(http) |
TSV |
| GSM7383490_matrix_saig_210928.mtx.gz |
70.4 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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