title: GSM5736036: NK MeDIP rep2; Mus musculus; MeDIP-Seq
library_construction_protocol: For sorting, spleens were dissociated and washed through 70um cell strainers and enriched with DX5+ MACS positive selection columns. For immune phenotyping and cell stimulation, spleens were dissociated and washed through 70um cell strainers. Lysates were lysed for red blood cells and resuspended in medium. Small intestinal ILC2 and ILC3 were isolated as previously described. Briefly, small intestines were dissected and washed with Peyer's patches and mesenteric fat removed. Intestines were opened lengthwise; gently agitated for 20 min in Hanks' balanced salt solution (HBSS) containing HEPES, bovine calf serum, and EDTA; and then vortexed. Small intestines were subjected to a second round of gentle agitation and vortexing in EDTA. The tissue was then rinsed with HBSS before digestion with collagenase IV (Sigma-Aldrich) in complete RPMI 1640 for 40 min at 37°C under agitation. Digests were filtered through 100-um mesh and subjected to density gradient centrifugation using 40 and 70% Percoll solutions. MeDIP-seq libraries were generated as previously described with few modifications. One hundred nanograms of sheared DNA was ligated with Illumina adapters, and methylation-enriched adapter-ligated DNA fragments were immunoprecipitated with 0.1 ug of anti-methylcytidine antibody (Eurogentec, BI-MECY-0100). MeDIP DNA fragments were amplified with Illumina barcodes with NEBNext High-Fidelity 2× PCR Master Mix (polymerase chain reaction) master mix (NEB, M0541). MeDIP-seq libraries were sequenced on Illumina NextSeq 500 platform. MRE-seq libraries were generated as previously described with few modifications. In brief, 20 ng of genomic DNA was digested by four restriction enzymes (HpaII, HinP1I, AciI, and HpyCH4IV) that generate a CG overhang. Adapter ligation was performed with custom Illumina adapters (5'-ACACTCTTTCCCTACACGACGCTCTTCCGATC*T-3' and 5'-P-CGAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3'). Adapter-ligated DNA fragments were amplified with Illumina barcodes with NEBNext High-Fidelity 2× PCR Master Mix master mix (NEB, M0541) and sequenced on Illumina NextSeq 500 platform. To identify 5hmC-enriched regions, we performed hmC-Seal on cell samples. In brief, 275 ng of sheared DNA was ligated with Illumina adapters. Hydroxymethylation-enriched adapter-ligated DNA fragments were glucosylated by incubating in a 50-ul solution containing 1× glucosylation buffer, 200 uM UDP-azide-glucose (Active Motif, 55020), and 5 U of T4 B-glucosyltransferase (Thermo Fisher Scientific, EO0831) at 37°C for 1 hour. After glucosylation, the DBCO-PEG4-biotin reaction and streptavidin C1 bead pull-down were same as the Nano-hmC-Seal. The beads were washed 10 times with 1× binding-washing buffer and twice with double-distilled water (ddH2O) and were resuspended in 15 ul of ddH2O. The captured DNA fragments were amplified and barcoded with Illumina barcodes with the NEBNext High-Fidelity 2× PCR Master Mix (NEB, M0541). hmC-Seal libraries were sequenced on Illumina NextSeq 500 platform.