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Low-input bulk RNA sequencing of thymic, mediastinal adipose tissue, and aortic arch CD4+CD8+ (DP) cells

ABSTRACT: Low-input bulk RNA sequencing of thymic, mediastinal adipose tissue, and aortic arch CD4+CD8+ (DP) cells

PROVIDER: PRJNA748740 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR15209551.fastq.gz	Fastqsanger.gz
	SRR15209552.fastq.gz	Fastqsanger.gz
	SRR15209553.fastq.gz	Fastqsanger.gz
	SRR15209554.fastq.gz	Fastqsanger.gz
	SRR15209555.fastq.gz	Fastqsanger.gz

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Low-input bulk RNA sequencing of thymic, mediastinal adipose tissue, and aortic arch CD4+CD8+ (DP) cells

Project description:Purpose: To establish if and how mediastinal and adipose tissue and aortic arch CD4+CD8+TCRb- cells (DPs) would differ from thymic DPs, we sorted DPs from all three regions by flow cytometry and subjected the cells to a low-input deep transcriptional profiling assay. Methods: Thymi, mediastinal adipose tissue, and aortic arches of five female, 8-week-old C57BL/6J mice were collected and pooled. Aortic arches and mediastinal adipose tissue were digested as previously described (PMID: 14597735) and single cell suspensions were generated from all three tissues. Four pools were generated per tissue. 400 viable CD4+CD8b+TCRb- cells were sorted into 8ul low-input lysis buffer containing RNaseH inhibitor, dNTPs, and 0.1% Triton X-100 in replicates. Material of 200 cells was reverse transcribed and amplified. Illumina libraries were prepared and sequenced. Results: A total of 2147 genes were differentially regulated between the different DP populations. Extrathymic DPs had 858 genes upregulated and 1284 genes downregulated compared to thymic DPs. 94 genes were commonly regulated in the two extrathymic DP subsets, whereas 23 genes were uniquely expressed in MAT DPs, 31 different genes were exclusively expressed in aortic arch DPs. Conclusion: Our study is the first to identify and provide deep transcriptional analysis two extra-thymic DP populations in mice.

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Project description:Aortic arch profiling of Ldlr-/- huCETP transgenic mice fed a low fat, cholesterol-containing western diet (LFWD, 9% fat, 0.15% cholesterol) starting at 8 weeks of age for 4, 8, 12, and 16 weeks. Ldlr-/- huCETP transgenic mice were fed a low fat, cholesterol-containing western diet (LFWD, 9% fat, 0.15% cholesterol) starting at 8 weeks of age for 4, 8, 12, and 16 weeks (Supplementary Figure 1). At each time point, 10 mice were studied. Mice were euthanized at each time point and the aortic arch of each mouse was immediately collected and flash-frozen in liquid N2. Mice were fasted overnight prior to euthanasia. the aortic arches of 10 mice in the same study group were pooled for profiling, resulting in one pool per study group. Tissues were homogenized and total RNA was extracted. The control RNA pool was composed of equal aliquots of RNA derived from the 4 pools (40 samples) from the LFWD 4-, 8-, 12- and 16-week time points.

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