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Patch-Seq of seven 5-HT neuronal subpopulations in zebrafish whole brain

ABSTRACT: Patch-Seq of seven 5-HT neuronal subpopulations in zebrafish whole brain

PROVIDER: PRJNA1160211 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR30644308_1.fastq.gz	Fastqsanger.gz
	SRR30644308_2.fastq.gz	Fastqsanger.gz
	SRR30644309_1.fastq.gz	Fastqsanger.gz
	SRR30644309_2.fastq.gz	Fastqsanger.gz
	SRR30644310_1.fastq.gz	Fastqsanger.gz

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Patch-Seq of seven 5-HT neuronal subpopulations in zebrafish whole brain

Project description:The goals of this study is to compare transcriptome profiles (RNA-seq) of seven 5-HT neuronal subpopulations in zebrafish whole brain. Tg(tph2:GFP) fish at 7 dpf were fixed in a recording chamber containing extracellular solution. The somata of 5-HT neurons from seven subpopulations (PT, SR-R, SR-M, SR-C, IR-R, IR-C, MO) were visualized under a fluorescence microscopy, then targeted and collected separately using glass electrode. For each sample, 10-12 neurons were collected and transferred to lysis buffer solution. Total RNA was extracted for construction of cDNA libraries, involving fragmentation, PCR enrichment, N7 index linking, and purification. A total of 71 QuantSeq libraries (N=9-12 samples for each subpopulation) were prepared using the Lexogen’s QuantSeq 3′ mRNA-Seq Library Prep Kit for Illumina according to the manufacturer’s instructions. Sequencing was carried out using Illumina NovaSeq 6000 with 150 bp paired-end reads. Gene expression level analysis was performed using R (Version 4.3.1) package Seurat (Version 5.0.2), and the mean gene expression matrix of all samples was obtained after processing by NormalizeData function. For marker gene analysis of each subpopulation, FindAllMarkers function in Seurat was used, with parameters set as min.pct=0.1 and logfc.threshold=0.25. Differential expression analysis revealed that each subpopulation expressed a set of featured genes, including specific markers for neurotransmitters, axon projection, neuron development, synapse, and ion channels, suggesting that seven 5-HT neuronal subpopulations display distinct genetic profiles. We demonstrated that seven 5-HT neuronal subpopulations are molecularly discriminative by expressing distinct molecular markers.

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Project description:Purpose: compare the gene expression diffenrence between wildtype and Pten-deficient 5-HT neurons. Methods: 5-HT neurons cell body are collected from acut brain slice with patch electrodes. Pten-deficient 5-HT neurons are obtained from Pet1-Cre; Ptenflox/flox mice. Normal 5-HT neurons are obtained from littermate wildtype mice. Reverse transcription was performed using a SMARTer Ultra Low RNA Kit (Clontech, Japan). Single-cell cDNA was amplified using an Advantage 2 PCR Kit (Clontech, Japan). Purified cDNA from individual cells were sequenced using the Illumina HiSeq 2000 platform. Sequence reads were aligned to the mouse mm9 genome using TopHat. Differential expression analysis was performed using DESeq (Bioconductor) followed by Student t-test. Genes were considered differentially expressed if p<0.05. Results: The somatic compartments of individual YFP-labeled 5-HT neurons in acute brain slices were harvested for RNA sequencing. Each of the cells had 2 X 107 reads mapping back to the mouse genome when analyzed using the RUM mapping program. We found 859 genes (381 up-regulated, 478 down-regulated) were differentially expressed in Pten-deficient 5-HT neurons. Gene ontology of differentially expressed genes revealed the biological processes up-regulated and down-regulated in Pten-deficient 5-HT neurons. The absence of Pten in 5-HT neurons was associated with significantly increased expression of genes involved in the regulation of axonal extension and cell size, cell morphogenesis and cytoskeleton organization

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