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

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.