title: Direct 5hmC immunoprecipitation (hMeDIP) sequencing from mouse cerebellum (input)
design: High molecular weight genomic DNA was extracted from these cerebella using the QIAGEN MagAttract HMW DNA Kit (QIAGEN, 67563) using a modified protocol available from the Oxford Nanopore Technologies Community website (Oxford Nanopore Technologies, 2022). DNA 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 then 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 Meehan (2014). 1 g sonicated DNA was incubated overnight at 4C rotating in IP buffer (10 mM Na-phosphate pH 7.0, 140 mM NaCl, 0.05% Triton X-100) with 2 L of either whole serum -5hmC antibody (ActiveMotif, 39769) or IgG-purified -5hmC antibody (ActiveMotif, 39791). Samples were then incubated with 40 L equilibrated Dynabeads Protein G (Invitrogen, 10003D) for 1 hour while rotating at 4C 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 L 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.