C19ORF84 connects piRNA and DNA methylation machineries to defend the mammalian germline
C19ORF84 connects piRNA and DNA methylation machineries to defend the mammalian germline
FACS purified P14 spermatogonial stem cells DNA from spermatogonia was isolated by proteinase K digest (10 mM Tris-HCl pH 8, 5 mM EDTA , 1% SDS, 0.3 M Na-acetate, 0.2 mg/ml proteinase K) overnight, followed by 2x phenol/chloroform/isoamylalcohol (25:24:1, Sigma-Aldrich) extraction and 1x chloroform extraction. The DNA was precipitated at -20˚C after addition of 1/10 volume 3M Na-acetate, 10µg linear acrylamide (Invitrogen) and 1 volume of isopropanol, washed 2x and solubilized in 5mM Tris pH8.  Methyl-seq libraries were prepared using the NEBnext Enzymatic Methyl-seq kit (NEB) according to the manufacturer's instructions