title: GSM2109922: 38_pronucleus_w-o-inh-undef sex; Mus musculus; OTHER
library_construction_protocol: Nuclei were fixed in separate drops of M2 (not containing BSA) with 2% formaldehyde for 15 minutes. Nuclei were washed through drops of complete M2. For zygote experiments without pronuclear extraction, zygotes were treated identically to oocytes. Here nuclei were separated into individual wells after SDS treatment and kept separate in the subsequent steps of the protocol. After fixation, zygotes or single isolated pronuclei were incubated in 9 μl of lysis buffer for at least 15 minutes on ice. Similarly zygotes or pronuclei were incubated in 9 μl of 1× NEB3 buffer supplemented with 0.6% SDS for 2 hours at 37° with shaking in humidified atmosphere. Then nuclei were incubated in 1× DpnII buffer (NEB) with 1× BSA and  5 U DpnII (NEB). After overnight incubation in a humidified incubator at 37° the DNA ends were ligated in 9 μl of 1× T4 DNA ligase buffer containing 5 U T4 DNA ligase (Thermo Scientific) and they were incubated at 16°C for 4.5 hours with 50 rpm rotation, and then were kept for 30 min at room temperature. DNA from each nucleus was amplified using illustra GenomiPhi v2 DNA amplification kit (GE Healthcare) or illustra Single Cell GenomiPhi DNA amplification kit according to manufacturer's recommendations, but both protocols were modified to include a decrosslinking step of an overnight (14-16 hours) incubation at 65°C. After the whole-genome amplification procedure, the DNA was purified. 1 μg (or all available amount, if the yield was lower) of DNA was diluted to 500 μl with sonication buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 0.1% SDS) and was sonicated using Branson Sonifier 150 for 20 seconds with power setting 3. The sonicated DNA was purified and then submitted for library preparation and sequencing. The library preparation was done by the VBCF NGS unit (vbcf.ac.at) following the standard DNA sample  preparation protocol for Solexa platforms (NEB reagents).
attributes.tag: GEO Accession
attributes.value: GSM2109922