GSM6303801: VASA_clone74_E4_dP_IG_DMR; Mus musculus; OTHER
DNA was isolated by lysing cells in lysis buffer (0.1M Tris buffer, 0.2M NaCl, 0.005M EDTA, 0.2% SDS) with 10mg/ml Proteinease K at 55°C, precipitated with Iso-Propanol, washed with 70% Ethanol and resuspended in TE buffer. Bisulfite conversion of DNA was established using the EZ DNA Methylation-Lightning Kit (Zymo Research cat#D5031). Converted DNA samples were subjected to an End repair reaction containing End repair mix and buffer (NEB cat#E6050) and 0.2-150ng DNA. DNA was purified using 2.5X SPRI Agencourt AMPure XP beads (Beckman Coulter cat#A63881). The eluted product was next subjected to an A-tail reaction including 10mM dATPs and Klenow Fragment 3´5´ exo- (NEB cat#M0212). DNA was purified with 2.5X SPRI beads. The clean DNA was tagged with an index oligo adapter in a ligation reaction using the Quick ligase Kit (NEB cat#M2200). Tagged products were then cleaned using 1.3X SPRI beads, and amplified for library preparation with 14 PCR cycles using the KAPA HiFi HotStart Ready Mix kit (Kapa Biosystems cat#KK2601), following the manufacturer's protocol. The reaction mix was then cleaned with 0.7X beads. Final libraries were pooled and sequenced on an Illumina NextSeq system using the 150-bp high-output sequencing kit.