GSM4975270: Blood mononuclear cells - cow 3- high - control; Bos taurus; Bisulfite-Seq
Cells were homogenized in 1 mL of Trizol reagent and DNA was extracted following the manufacturer's instructions. For each sample, 40-50ng of DNA was digested for 2 hours with MSP1 at 20U/sample at 37C, followed by 2 hours with TaqI/sample at 65C. Digested DNA was size selected with <300 bp DNA fragments and then bisulfite-treated with the Methylamp DNA Biulfite Conversion Kit (Epigentek Cat. # P-1001). Conversion efficiency of bisulfite treated DNA was determined by real time PCR using two primer pairs with using control DNA. A method of reduced representation bisulfite sequencing (RRBS), Enhanced MethylSeq (Epigentek, NY), was used to assess DNA methylation in control and HS samples. Libraries were prepared from 2-10ng of genomic DNA using the PBAT parameter (Post-Bisulfite Adapter Tagging. Libraries were prepared by DNA end polishing and adaptor ligation followed by library amplification using indexed primers and library purification. Libraries were sequenced on an Illumina HiSeq 4000 with single reads SR50 (Epigentec, NY (https://www.epigentek.com).