GSM7173861: input_blood_female_DEHP_4; Mus musculus; OTHER
The AllPrep DNA/RNA/miRNA Universal Kit (Qiagen, Cat. #80224) was used to extract DNA from blood and cortex tissue For each sample, a total of 750 ng of genomic DNA was sheared by adaptive focused acoustics, using the Covaris S220 (Covaris). This sheared DNA was blunt-ended and phosphorylated. A single A-nucleotide was then added to the 3'-end of the fragments in preparation for ligation of adapter duplex with a T overhang. The ligated fragments were cleaned using Qiagen's MinElute PCR purification columns. DNA standards for hMeDIP-seq (Diagenode, 5-hmC, 5-mC, & cytosine DNA standard pack for hMeDIP, cat # AF-107-0040) were added to each sample before denaturation and resuspension in ice-cold immunoprecipitation buffer (10 mM Sodium Phosphate pH 7.0, 140 mM NaCl, 0.05% Triton X-100). A 10% volume was input before 2 µg of a 5-hmC-specific antibody (Active Motif, Cat # 39791) was added for immunoprecipitation overnight at 4°C with rotation. Dynabeads Protein-G (Invitrogen) were added to the immunoprecipitation to perform the pull-down of 5-hmC-enriched fragments. The 5-hmC-enriched DNA fragments were then released from the antibody by digestion with Proteinase K (Ambion).The libraries were pooled and then sequenced on a NovaSeq6000 instrument.