GSE168209
Genome-wide methylation analysis of murine innate lymphoid cells
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. Overall design: Examination of murine NK cells, ILC1, ILC2, ILC3 and LTi cells from lymph nodes.