GSM7818118: PoolA_DBA_Lamin_TwoCell_rep3; Mus musculus; OTHER
Zona pellucida was removed by treatment with 0.5% pronase in M2 at 37 °C. Polar bodies were separated from the embryos by gentle pipetting after trypsin treatment and discarded. For each replicate, a pool of 10-20 blastomeres (10 to 20 zygotes or 5 to 10 2-cell embryos) were collected in 2 μL DamID buffer (10 mM TRIS acetate pH 7.5, 10 mM magnesium acetate, 50 mM potassium acetate) and stored at -80 °C until downstream processing. Approximately 300 ng of PCR purified DNA was end-repaired using an End-It DNA End-Repair Kit. The blunt, phosphorylated ends were treated with Klenow fragment (exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3' end. Between steps, the DNA was purified with Agencourt AMPure XP beads with a 1.8 x bead volume and a 1.0 x bead volume after the ligation reaction. After adapter ligation, DNA was PCR amplified with Illumina primers for 8 cycles.