Summary: The goal of this study was to identify distinct cell populations that arise from ventral spinal cord pMN progenitors. To do so, we sorted fluorescently marked pMN cells obtained from Tg(olig2:EGFP) zebrafish embryos at 24, 36 and 48 hours post fertilization and performed 10X Chromium single cell RNA-seq.
Overall Design: Three samples obtained from wild-type zebrafish embryos at three developmental timepoints were analyzed.
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Growth Protocol: | developmental stage: 24 hours post fertilization; strain: Tg(olig2:EGFP) ZFIN ID: ZDB-ALT-041129-8; developmental stage: 36 hours post fertilization;strain: Tg(olig2:EGFP) ZFIN ID: ZDB-ALT-041129-8; developmental stage: 48 hours post fertilization; strain: Tg(olig2:EGFP) ZFIN ID: ZDB-ALT-041129-8 |
Treatment Protocol: | - |
Extract Protocol: | 24, 36, and 48 hpf Tg(olig2:EGFP) euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 μl of pre-chilled Ca free Ringers solution (116 mM NaCl, 2.6 mM KCl, 5 mM HEPES, pH 7.0) on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 μl of protease solution (10 mg/ml BI protease, 125 U/ml DNase, 2.5 mM EDTA, 1X PBS) was added to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 μl of STOP solution (30% FBS, 0.8 mM CaCl2, 1X PBS) was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4°C and supernatant was removed. On ice, 1 ml of chilled suspension media (1% FBS, 0.8 mM CaCl2, 50 U/ml Penicillin, 0.05 mg/ml Streptomycin) was added to samples and then spun down again at 400g for 5 min at 4°C. Supernatant was removed, and 400 μl of chilled suspension media was added and solution was filtered through a 35 μm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU-SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 μl of 1X PBS. |
Library Construction Protocol: | The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell 3' Reagent Kit part no. PN-1000075. Libraries were sequenced on the Illumina NovaSEQ6000 Instrument. |
Molecule Type: | poly(A)+ RNA |
Library Source: | |
Library Layout: | PAIRED |
Library Strand: | Forward |
Platform: | ILLUMINA |
Instrument Model: | Illumina NovaSeq 6000 |
Strand-Specific: | Specific |
Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
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