Gene Expression Nebulas
A data portal of transcriptomic profiles analyzed by a unified pipeline across multiple species

Gene Expression Nebulas

A data portal of transcriptome profiles across multiple species

PRJNA656271: Single cell RNA-seq analysis of pMN neural progenitors from zebrafish

Source: NCBI / GSE155988
Submission Date: Aug 10 2020
Release Date: Aug 11 2020
Update Date: Aug 19 2020

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.

GEN Datasets:
GEND000146
Strategy:
Species:
Healthy Condition:
Cell Type:
Development Stage:
Protocol
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. 
Sequencing
Molecule Type: poly(A)+ RNA
Library Source:
Library Layout: PAIRED
Library Strand: Forward
Platform: ILLUMINA
Instrument Model: Illumina NovaSeq 6000
Strand-Specific: Specific
Samples
Basic Information:
Sample Characteristic:
Biological Condition:
Experimental Variables:
Protocol:
Sequencing:
Assessing Quality:
Analysis:
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
Publications
Prdm8 regulates pMN progenitor specification for motor neuron and oligodendrocyte fates by modulating the Shh signaling response.
Development (Cambridge, England) . 2020-08-27 [PMID: 32680935]