title: GSM6341106: Sperm, FCD239C; Mus musculus; Bisulfite-Seq
library_construction_protocol: Frozen sperm/isolated spermatogonia were thawed on ice and genomic DNA isolation was performed using Qiagen DNeasy Blood & Tissue Kits (Qiagen, Germany) according to the manufacturer's guide, with modifications. For sperm samples, sperm was incubated overnight at 37oC in a buffer containing EDTA, Tris , sarkosyl , dithiothreitol (DTT) and proteinase K (Invitrogen, Canada). The following day, Buffer AL was added to the buffer, and incubated at 70oC for 10min and the manufacturer's guide was continued. Reduced representation bisulfite sequencing (RRBS) libraries were prepared according to previously published protocols using gel-free technique (Boyle, P. Genome Biol., 2012).  Briefly, ~500ng of  DNA was digested with MspI restriction enzyme, followed by size selection, gap filling and 3'-A-tailing.  Ligation of NEBNext methylated adaptors was performed, followed by bisulfite conversion and index PCR amplification.