title: GSM6743125: 2C_Ama_35h_3; Mus musculus; OTHER
library_construction_protocol: Early stage zygotes were collected and cultured until they reached the S-phase at each developmental stage, based on their time after hCG injection. Embryos were collected at different time points to achieve sampling over the entire S-phase. For the parthenogenetic embryos and IVF-derived zygotes, the timing of S-phase was calculated based on the time after activation and insemination, respectively. Zona pellucida was removed by exposure to acid Tyrode and each blastomere was dissociated by gentle pipetting after trypsin treatment. For the Repli-seq with physically isolated pronuclei, we distinguished maternal and paternal pronucleus based on their size and their relative position to the second polar body and isolated either of them using micromanipulation. The remaining zygote containing a single pronucleus was also collected after removal of polar body so that both pronuclei from the same zygote were further processed for Repli-seq. Individual blastomeres or pronuclei were placed into 8-strip PCR tubes containing lysis buffer and extracted DNA was fragmented by heat incubation. Fragmented DNA was tagged by the universal primer (5'-TGTGTTGGGTGTGTTTGGKKKKKKKKKKNN-3') and amplified with WGA primer sets which have individual barcode. Amplified DNA was purified by the QIAquick 96 PCR Purification Kit (QIAGEN) and concentration was determined by the NanoDrop (Thermo Scientific). Equal amount of DNA from each samples (up to 96 samples) were pooled and 1ug of them was ligated with the illumina adaptors using the NEBNext Ultra II DNA Library Prep Kit (NEB). Illumina sequence (NEBNext Multiplex Oligos for Illumina; NEB) were added to the adaptor- ligated samples by the PCR. Clean up and size selection of the PCR product was done using SPRIselect (Beckman Coulter) and the quality of the library was confirmed by 2100 Bioanalyzer with the High Sensitivity DNA Kit (Agilent).