Unknown,Transcriptomics,Genomics,Proteomics

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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: 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.

INSTRUMENT(S): NextSeq 500

ORGANISM(S): Mus musculus

SUBMITTER: Andreas Diefenbach

PROVIDER: E-MTAB-8883 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare adhesion gene clusters and clock genes / biomarkers at different time points throughout the day in sorted murine lympahtic endothelial cells. Methods: RNA of sorted dermal LECs harvested in TriZol-LS was purified using Direct-zol RNA mini Prep Kit (ZymoResearch) following manufacturer’s instructions. Isolated RNA was quantified using the Nanodrop (Thermofisher) and analyzed on a Bioanalyzer (Agilent) using the RNA 6000 Nano or Pico Kit (Agilent). 75 ng of eluted total RNA was digested with DNase (Thermofisher) to remove DNA contaminations. An additional purification step with RNAClean XP Beads (Agencourt) was performed. The purified, bead-bound RNA was directly used as input in the SMART-Seq v4 Ultra Low Input RNA Kit (TaKaRA Bio). Full-length cDNA was generated following manufacturer’s instructions. Full-length cDNA was quantified using the Qubit dsDNA HS Assay Kit (Invitrogen) with the Qubit fluorometer. Finally, sequencing libraries were generated using 500 pg full-length cDNA following the NexteraXT protocol (Illumina). Libraries were quantified on a Bioanalyzer (Agilent) using the DNA 1000 Kit (Agilent) and sequenced on a HiSeq1500 system (Illumina) with a readlength of 100 nt, single-end mode. Results: obtained transcriptome profiles were processed on a Galaxy web interface hosted by LAFUGA (Gene Center, Munich). After demultiplexing and trimming data was mapped against the mouse genome (mm10) using RNA-STAR mapper (Galaxy Version 2.5.2b-0). Abundant reads were counted using HTSeq-count (Galaxy Version 1.0.0). Afterwards, gene expression analysis to detect differentially expressed genes was performed using DESeq2 (Galaxy Version 2.11.40.6), setting the false discovery rate (FDR) <0.05. Comparing the expression profiles and the enrichment of gene ontology cluster adhesion revealed a highly time-of-day dependent expression profile of genes. Conclusions: Our study represents the first detailed analysis of time-of-day dependent lymphatic endothelial cell transcriptomes, with respect to adhesion and clock genes. This will provide valid input into chronotherapy, timed delivery of substances and new therapeutic avenues.

Dataset Information

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

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