GSM6450978: Sperm, Control1, 18month [CC33-3]; Homo sapiens; OTHER
Frozen sperm was thawed on ice and genomic DNA isolation was performed using Qiagen DNeasy Blood & Tissue Kits (Qiagen, Canada) according to the manufacturer's guide, with modifications. For sample  were 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. Using 1ug of DNA from each sample, whole genome bisulfite sequencing (WGBS) library preparation and targeting bisulfite sequencing was libraries were performed as described previously. Briefly, DNA was sonicated to obtain 300-400bp fragments, proceeded by DNA-end repair, 3'-end adenylation, adaptor ligation and clean up, according to the KAPA High Throughput Library Preparation kit protocol (Roche/KAPA Biosystems). Samples were then bisulfite converted using the EpiTect Fast DNA bisulfite kit (Qiagen) followed by 9-12 cycles of PCR amplification. The final WGBS libraries were purified using Agencourt AMPure Beads (Beckman Coulter) and were then enriched using our human sperm specific panel, using the SeqCap Epi Enrichment System protocol developed and optimized by Roche NimbleGen. Equal amounts of the multiplexed WGBS libraries (84ng each) were combined and hybridized to the human sperm specific capture panel at 47C for 72h. The captured libraries were washed, recovered and PCR amplified before final purification was conducted. The final capture libraries were sequenced one a single lane of the Illumina HiSeq4000 system using 100-bp paired end sequencing.