hMeDIP-seq of mouse cerebellum using nanopore sequencing

5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are modified versions of cytosine in DNA with roles in regulating gene expression. Using whole genomic DNA from mouse cerebellum, we benchmark 5mC and 5hmC detection by Oxford Nanopore Technologies sequencing against other standard techniques. In addition, we assess the ability of duplex base-calling to study strand asymmetric modification. Nanopore detection of 5mC and 5hmC is accurate relative to compared techniques and opens new means of studying these modifications. Strand asymmetric modification is widespread across the genome but reduced at imprinting control regions and CTCF binding sites in mouse cerebellum. Here we demonstrate the unique ability of nanopore sequencing to improve the resolution and detail of cytosine modification mapping. DNA from mouse cerebellum was sheared to a target of 6 kb using a Covaris g-TUBE (Covaris, 520079). An aliquot was sequenced as a control on an R10.4.1 MinION flow cell (Oxford Nanopore Technologies, FLO-MIN114) using Ligation Sequencing (SQK-LSK114) Kit 14 following the associated protocol. Sheared DNA was sonicated in triplicate using a Bioruptor® Pico sonication device (Diagenode, B0106001) in a 1.5 mL Bioruptor® Pico Microtube with cap (Diagenode, C30010016) to target fragment lengths < 500bp. A 2200 TapeStation system (Agilent, G2991A) with D1000 ScreenTapes (Agilent, 5067-5582) and associated reagents (Agilent, 5067-5583) was used to assess the fragment size distribution of each replicate (mean 362 bp). 5hmC immunoprecipitation was performed based on Nestor and Meehan23. 1 μg sonicated dsDNA was incubated overnight at 4°C rotating in IP buffer (10 mM Na-phosphate pH 7.0, 140 mM NaCl, 0.05% Triton X-100) with 2 μL of either whole serum α-5hmC antibody (ActiveMotif, 39769) or IgG-purified α-5hmC antibody (ActiveMotif, 39791). Samples were then incubated with 40 μL equilibrated Dynabeads Protein G (Invitrogen, 10003D) for 1 hour while rotating at 4°C and washed three times in ice-cold IP buffer for 5 minutes at room temperature. Bound DNA was eluted in digestion buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 0.5% SDS) and 200 ng proteinase K (ThermoFisher Scientific, EO0491) for 3 hours at 50 °C on a shaker at 800 rpm. Immunoprecipitated DNA was purified using the QIAQuick PCR Purification kit (QIAGEN, 28104) following manufacturer instructions and eluted into 50 μL elution buffer before sequencing. The eluate was prepared for sequencing following the MinION protocol SQK-LSK114. Libraries were loaded onto three R10.4.1 MinION flow cells and sequenced, generating 154-382 Mb of sequence data per flow cell.