GSM7988976: Y3_liver_240_Input; Mus musculus; OTHER
Genomic DNA was isolated from ~25 mg of frozen tissue using the Quick-DNA Miniprep Pus Kit (Zymo Research) following the manufacturer's protocol with an overnight proteinase K digestion. The amount and quality of extracted DNA was assessed by NanoDrop One (Thermo Fisher). The hMeDIP assay was performed using the MagMeDIP-seq Package (Diagenode, C02010040) with an antibody against 5hmC (Diagenode, C15200200-50). 1.2 µg of genomic DNA was sonicated into ~200 bp fragments using the S220 focused ultrasonicator (Covaris). To maximize IP yield, we processed the samples in duplicates and then pool before performing IPure purification. Prior to immunoprecipitation, samples were spiked with hydroxymethylated (Diagenode, C02040018) and unmethylated DNA controls. Immunoprecipitated and 10% input DNA was used to prepare libraries according to the MagMeDIP-seq Package protocol except libraries were dual indexed using the NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs) (New England Biolabs) followed by PCR amplification. The quantity and quality of individual libraries were verified on a BioAnalyzer DNA 1000 (Agilent) then pooled into equimolar amounts. The quantity of the pooled library was assessed using the NEBNext Library Quant Kit (New England Biolabs) and Qubit HS DNA kit (Thermo Fisher).