GSM1366370: Oocyte_1; Mus musculus; Bisulfite-Seq
Approximately 100-130 pronuclei in each treatment were harvested, and lysed in 5  μl lysis buffer (20 mM Tris, 2 mM EDTA, 20 mM KCl, 0.3% Triton-X 100, 1 mg/ml QIAGEN protease) at 50°C for 3h. Protease was inactivated at 75°C for 30 min. Genomic DNA from pronuclei was digested by 10 U TaqI (NEB) for 3 h at 65°C. Then the digested DNA was end-repaired and dA-tailed with exonuclease-deficient Klenow Fragment (Fermentas) in the presence of 40 μM dATP, 4 μM dGTP and 4 μM dCTP. Next the Illumina TruSeq adapters were ligated to the DNA fragments with concentrated T4 DNA ligase (Fermentas) overnight. After that, bisulfite conversion of the library was performed using MethylCode DNA Methylation Kit (Invitrogen). Then bisulfite-converted DNA was amplified by 20 cycles of PCR using PfuTurboCx polymerase (Agilent Technologies), and purified by AMPure XP beads (Beckman). Finally an extra round of PCR amplification was performed to increase the amount of DNA to about 500 ng with Phusion HF Master Mix (NEB), and the 200~500 bp DNA fragments were selected on a 12% polyacrylamide gel. The size distribution of the final library was analyzed by Fragment Analyzer (Advanced Analytical Technologies) and the libraries were sequenced on Illumina HiSeq 2000 or 2500.
GEO Accession
GSM1366370