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RNA sequencing of primary isolated unstimulated murine splenic DC1 and DC2 from specific pathogen free (SPF), Germ free (GF) and Interferon alpha/beta receptor 1 (Ifnar) knock out mice

ABSTRACT: RNA sequencing of primary isolated unstimulated murine splenic DC1 and DC2 from specific pathogen free (SPF), Germ free (GF) and Interferon alpha/beta receptor 1 (Ifnar) knock out mice

PROVIDER: PRJEB37204 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR3987200_1.fastq.gz	Fastqsanger.gz
	ERR3987200_2.fastq.gz	Fastqsanger.gz
	ERR3987210_1.fastq.gz	Fastqsanger.gz
	ERR3987210_2.fastq.gz	Fastqsanger.gz
	ERR3987201_1.fastq.gz	Fastqsanger.gz

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RNA sequencing of primary isolated unstimulated murine splenic DC1 and DC2 from specific pathogen free (SPF), Germ free (GF) and Interferon alpha/beta receptor 1 (Ifnar) knock out mice

Project description:The transcriptomes of primary isolated unstimulated murine splenic cDC1 and cDC2 from specific pathogen free (SPF) were compared to cDC1 and cDC2 from Germ Free (GF) and Interferon alpha/beta receptor 1 (Ifnar) knock out mice. Total RNA was prepared from sort-purified cDC1 and cDC2 (for each sample 3 individual mice were pooled) using Trizol reagent (Invitrogen) in combination with the miRNeasy Micro Kit (Qiagen) according to the manufacturer’s instructions. For poly-A-dependent cDNA synthesis and a first amplification step 10 ng of total RNA was used as input in the Smart-Seq v4 mRNA Ultra Low Input RNA Kit (Clontech) and processed according to the manufacturer’s instructions. 1 ng of the purified cDNA was used for tagmentation and library completion with the Nextera XT library preparation kit (Illumina). In the following, 2x75nt paired-end sequencing was performed on a NextSeq 500.

2020-03-30 | E-MTAB-8883 | biostudies-arrayexpress

RNA sequencing of primary isolated unstimulated murine splenic cDC from specific pathogen free (SPF), Germ Free (GF) and Interferon alpha/beta receptor 1 (Ifnar) knock out mice.

Project description:The transcriptome of primary isolated unstimulated murine splenic cDC from specific pathogen free (SPF) was compared to cDC from Germ Free (GF) and Interferon alpha/beta receptor 1 (Ifnar) knock out mice. Cells were isolated and sorted by flow cytometry to high purity. Total RNA was isolated using the RNeasy plus micro kit (Qiagen). Barcoded mRNA seq libraries were prepared from 100 ng of total RNA (RIN>8) using a NEBnext poly(A) mRNA Magnetic Isolation Module and NEBnext UltraII lib prep kit for Illumina (New England Biosciences) according to the manual. Single-end RNA sequencing (59nt) was performed on a HiSeq 2500 (Illumina). Barcoded RNA-seq libraries were onboard clustered using HiSeq® Rapid SR Cluster Kit v2 using 8pM and 59bps were sequenced on the Illumina HiSeq 2500 using HiSeq Rapid SBS Kit v2 (59 Cycle). The raw output data of the HiSeq was preprocessed according to the Illumina standard protocol.

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Project description:While conventional dendritic cells (DC)1 and DC2 are found in tumors, DC1 were shown to control tumor response to checkpoint blockade in preclinical models and are associated with better overall survival in cancer patients, reflecting their specialized ability to prime CD8+ T cell responses. Paradoxically, DC1 can also be found in tumors that resist checkpoint blockade, suggesting that, like many tumor infiltrating T cells, DC1 functionality may be altered in tumors. To address this question, we performed scRNAseq analyses to characterize DC gene architecture in mouse non-small cell lung cancer (NSCLC) lesions. To understand the relationship of the resulting transcriptional signatures with antigen uptake and migration, we profiled lung DCs in CCR7-/- mice, or DCs that had taken up (i) GFP from GFP-expressing tumors that had been seeded in the lung, and (ii) fluorescent microbeads that had been applied intranasally.

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