GSM9006207: sperm, TBT 10nM; Bos taurus; Bisulfite-Seq
Genomic DNA was isolated from TBT exposed and unexposed sperm using a modified protocol from Quiagen Blood and Tissue Kit (60954). After DNA isolation, an Enzymatic Methyl seq Conversion kit module (NEB#E7125S) was used to convert unmethylated cytosines in genomic DNA (gDNA) into uracils, generating methylation-converted DNA for PCR amplification and downstream epigenomic analyses. For whole genome enzymatic methyl seq analysis (EMSeq), fragmentation of gDNA isolated from TBT exposed and unexposed sperm was performed on a M220 sonicator (Covaris). DNA fragments were end repaired/dA-tailed followed by adaptor ligation. A two-step enzymatic conversion protecting 5mC and 5hmC from deamination was performed prior to deamination of the cytosines residues. Fragments were PCR amplified to enrich libraries and incorporate barcodes. The final amplified cDNA constructs were purified by addition of Axyprep Mag PCR Clean-up beads (Axygen) selecting for an insert size of 550 bp.  Libraries were quantified with the Qubit HS DNA kit (Invitrogen) and the fragment size analyzed on a 5200 Fragment Analyzer (Agilent).  Libraries were diluted according to the standard protocol for sequencing on the NovaSeq 6000 (Illumina).