GSE168208
Next generation expression analysis of murine innate lymphoid cells
Purpose: Characterization of murine innate lymphoid cells (ILC) by next generation expression analysis Methods: Lymph node cells from Rorc(gt)-GFP reporter mice were independently sorted three times by flow cytometry to isolate ILC1, ILC2, ILC3 and lymphoid tissue inducer (LTi) cells. Subsequently RNA was prepared (Rneasy Micro Kit, Qiagen) and subjected to cDNA synthesis by using the SMART Seq v4 Ultra Low Input RNA Kit (Takara Bio) according to the manufacturer's protocol. Library preparation was performed with Nextera XT DNA Library Prep Kit (Illumina) and next generation sequencing was carried out on an Illumina HiSeq2500 using 50 bp single reads.  Adapter contamination were removed by trim_galore and cutadapt from the sequence reads that passed quality filters (Phred score > 30). Resulting libraries were aligned to the mouse reference genome (GRCm38) using the RNA-seq aligner STAR with default parametrization. Reads aligned to annotated genes were quantified with the HTseq-count program using gene annotations from Ensembl release 81. The determined read counts served as input to DESeq2 for pairwise detection and quantification of differential gene expression. For DESeq2 parametrization we used a beta prior and disabled the Cook distance cutoff filtering. Results: The sequencing depth of our libraries varies between 25.5 and 39.2 million reads. The mapping rates for the libraries were found in the range between 93.9 and 98.0 %. Unsupervised hierarchical clustering of the top 50 differentially expressed genes show a close relationship of the samples within a group and higher similarity between ILC3 and LTi cells whereas ILC1 and ILC2 appear as separated populations. Among the differentially expressed genes we identified already described transcripts like Gzma (ILC1), Klrg1 (ILC2) and Il22 (ILC3, LTi) as well as uncharacterized genes that may play a role for the ILC lineage and function in lymphoid organs. Conclusions: We report a detailed next generation transcriptome analysis of ILC populations from murine lymph nodes. Our analysis helps to define the developmental status of ILC in secondary lymphoid organs, which might represent a precursor state that can be further modulated in tissues. Overall design: Examination of murine ILC1, ILC2, ILC3 and LTi cells from lymph nodes.