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Genome-wide methylation analysis of murine innate lymphoid cells

ABSTRACT: Purpose: Identification of differentially methylated regions (DMRs) in murine innate lymphoid cells (ILC) Methods: Numerous preparations of lymph node cells from mice were independently sorted by flow cytometry to isolate Natural killer (NK) cells, ILC1, ILC2, ILC3 and lymphoid tissue inducer (LTi) cells. Genomic DNA from the sorted, fixed cells was extracted using the NucleoSpin Tissue kit (Macherey-Nagel), including a crosslink removal step that was described recently (Kyburz et al., J Allergy Clin Immunol 143, 1496-1512 e1411 (2019)). The resulting single-stranded DNA was converted with bisulfite using the EZ DNA Methylation-Direct Kit (Zymo Research) and fragmented by sonication (Covaris S220, 10% duty cycle, 175W peak incident power, intensity 5, 200 cycles per burst, 120 seconds). The fragmented DNA served as input for the Accel-NGS Methyl-Seq DNA Library Kit (Swift Biosciences) and resulted in libraries that were sequenced on an Illumina NovaSeq 6000 . After quality control, trimming and mapping by using BSMAP, we identified differentially methylated regions by pairwise comparison of the samples by using metilene. Results: The sequencing depth of our libraries varies between 210 and 282 million paired end reads. More than 98% of the murine CpG motifs were covered one time and 76% of the motifs at least five times. The pairwise comparison of the methylomes identified most of the differential methylated regions around the transcriptional start sites. Less differences were found between NK and ILC1 as well as between ILC3 and LTi cells. Among the DMRs showing meaningful differences we identified known demethylated regions in Tbx21, Ifng, Gata3, Il5 and Ccr6 as well as so far uncharacterized regions in Gpr18, Ptgir, Il22 or Il23r. Conclusions: Here we report for the first time a genome-wide methylation study on ILC subpopulations that will help to understand development and function of these cells.

ORGANISM(S): Mus musculus

PROVIDER: GSE168209 | GEO | 2021/05/31

REPOSITORIES: GEO

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Project description: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.

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Genome-wide methylation analysis of murine innate lymphoid cells

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