EMSeq_ZF_2025_26
We used Enzymatic Methyl-seq. We extracted DNA according to the manufacturers protocol using innuPREP DNA Mini Kit (Analytik Jena GmBH) from 3 uL (nucleated) red blood cells. Next-generation sequencing was outsourced to The Hospital for Sick Children (Toronto, Canada). DNA was quantified using a Qubit High Sensitivity Assay. Two hundred ng of DNA was used as input material for library preparation using the NEBNext Enzymatic Methyl-seq Kit following the manufacturer's instructions. Briefly, DNA was fragmented by sonication to an average of ~500bp using a Covaris LE220 instrument. Fragmented DNA was end-repaired and adapters were added by ligation. 5-methylcytosines and 5-hydroxymethylcytosines were oxidized by TET2 and cytosines were deaminated by APOBEC. Methyl-seq libraries were amplified by 5 PCR cycles. Libraries were run on a Bioanalyzer DNA High Sensitivity chip to check for size and absence of primer dimers, and quantified by qPCR using Kapa Library Quantification Illumina/ABI Prism kit. Validated libraries were pooled in equimolar quantities and sequenced to a minimum of ~100M paired-end reads per sample on 10B flow cells on the NovaSeqX platform (2x150bp).