MeDIP-Seq of Bubalus bubalis: Cloned and IVF embryos at 2-, 8-cell and blastocyst stage
Illumina library preparation was performed by using the NEBNext Ultra DNA Library Prep Kit for Illumina (NEB, New England Biolabs Inc., USA #E7370S). Sonicated DNA (25ng from each pool) was end repaired into dA-tailed fragments. Then the NEBNext-indexed adaptor was ligated into sonicated DNA fragments. Adaptor-ligated DNA was further cleaned up by AMPure XP beads (Beckman Coulter, Inc., Life Sciences, United States #A63881) and then subjected to the MeDIP procedure. The Input DNA was linked to different index primers. After immunoprecipitation, adaptor-ligated DNA was extracted by phenol/chloroform and precipitated by ethanol. The antibody-enriched DNA was subsequently amplified by using an index primer and universal PCR primer provided in NEB Multiplex Oligos for Illumina. PCR was performed with 20 cycles for each MeDIP sample. The PCR products were then purified by AMPure XP beads and eluted with TE buffer. The library concentration and size distribution was analyzed by Agilent 4200 Tapestation.The library was run on Agilent 4200 Tape Station and the resulting profile was evaluated.
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