hMeDIP-Seq of human: HCC827 cell line in hypoxia
A total of 1 g of genomic DNA (gDNA) was sheared by adaptive focused acoustics, using the Covaris S220 (Catalog #4,465,653; Covaris). This sheared DNA was next blunt-ended and phosphorylated. A single adenine nucleotide was then added to the 3 end of the fragments in preparation for the ligation of the adapter duplex with a thymine overhang. The ligated fragments were cleaned using Qiagens MinElute PCR purification columns (Catalog #28,004; Qiagen). DNA standards for HMeDIP (Diagenode 5-hmC, 5-mC, and cytosine DNA standard pack for HMeDIP; Catalog #AF-107-0040) were added to each sample before denaturation. Resuspension was then performed in ice-cold immunoprecipitation buffer (10mM sodium phosphate pH 7.0, 140mM sodium chloride (NaCl), 0.05% Triton X-100). A 10% volume (input) was retrieved before 2 g of a 5-hmCspecific antibody (Active Motif, Catalog # 39,791) was added for immunoprecipitation overnight at 4C with rotation. Dynabeads Protein-G (Catalog #10003D; Invitrogen) was used to pull down 5-hmCenriched fragments. The 5-hmC-enriched DNA fragments (from immunoprecipitation) were then released from the antibody by digestion with Proteinase K (Catalog #AM2548; Ambion). After cleanup with AMPure XP beads (Product #A63880; Beckman Coulter), the percentage input enrichment (%input) in the IP was evaluated by qPCR, using hydroxymethylated, methylated, and unmethylated primers for spike-ins. Samples with high %input for the 5-hmC spike-intypical inclusion threshold was >80%were then PCR amplified for the final library production, cleaned using AMPure XP beads, and quantified using the Qubit assay (Catalog #Q32850; Invitrogen) and TapeStation High Sensitivity D1000 kit (Catalog #G2991AA; Agilent). Single-end, 50-bp reads were obtained for each library by sequencing on the HiSeq X ten system (Illumina).