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Genotyping was performed by PCR using the previously described primers for the G9a alleles (Tachibana et al., 2007) and GCAGAACCTGAAGATGTTCGCGAT and AGGTATCTCTGACCAGAGTCATCC for the Zp3-Cre transgene. JF1 mice were described previously (Takada et al., 2013). mice of 10 weeks or older were placed together with males in the same cage. Vaginal plugs were checked the next morning. Males were removed after a plug was observed and females were maintained for 20 days, 2000. ,
, FGOs were harvested from P15 or adult (10 weeks or older) ovaries by needle puncture. Superovulation was induced by injecting females sequentially with pregnant mare serum gonadotrophin and human chorionic gonadotropin. Cumulus-oocyte complexes were collected from the oviducts and fertilized with C57BL/6J or JF1 sperm for assessment of preimplantation development, WGBS, and RNA-seq. MII oocytes were harvested by digestion in hyaluronidase (H4272, Sigma-Aldrich). Cumulus cells were removed by capillary washing in PBS and zygotes were cultured in EmbryoMax KSOM Medium (1X) w/ 1/2 Amino Acids (Merck Millipore) or KSOM AA (Ho et al., 1995) at 37 C and 5% CO 2 . Two-cell embryos were harvested within 2 hours after the completion of the first mitotic division for WGBS and at 24 hours for RNA-seq, Oocyte Collection, IVF and Embryo Culture GOs were harvested by sequential digestion of P10 ovaries in 1 mg/ml collagenase, vol.5147, pp.34-10533
Total RNA was extracted from pooled oocytes using Trizol reagent (Thermo Fisher Scientific). cDNA was synthesized using PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara Bio Inc.) and subjected to PCR using KAPA HiFi HotStart ReadyMix PCR Kit (Kapa Biosystems) and TaKaRa PCR Thermal Cycler Diceâ ,
Proteins were separated on a 12%-15% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane by wet blotting. The blot was blocked by 5% skimmed milk or 5% BSA in TBS with Tween-20 ,
Signals were detected using Chemi-Lumi One Ultra (Nacalai Tesque) on ImageQuant LAS4000 mini (GE Healthcare Life Sciences). Can Get Signal Immunoreaction Enhancer Solution (TOYOBO) was used to detect H3K9me2 ,
, Ovary Sectioning and Hematoxylin-Eosin Staining Ovaries were embedded in OCT and flash frozen. Frozen sections were prepared using CM3050 S Research Cryostat (Leica)
, BioAcademia) were used. Alexa Flour 488-, 555-, or 647-conjugated IgG (Thermo Fisher Scientific) was used as the secondary antibody. Oocytes and embryos were mounted in VECTASHEILD medium with DAPI (Vector Laboratory) and observed under LSM700 confocal laser scanning microscope (Carl Zeiss). The different anti-H3K9me2 antibodies displayed similar results, Active motif), H3K9me3 (MABI0318, MBL), CDX2 (ab157524, abcam), SOX2 (ab92494, abcam), SOX17 (AF1924, R&D Systems), and HP1b, vol.39239, pp.70-223, 2012.
Zona pellucida was removed by acid Tyrode's solution. ChIP was performed with anti-H3K9me2 antibody (abcam ab1220) in 200 mL NChIP buffer. WGBS libraries were sequenced on HiSeq 1500 or HiSeq 2500 (Illumina) (FGOs and blastocysts: HCS v2.0.5 and RTA v1.17.20; MII oocytes and 2-cell embryos: HCS v2.2.68 and RTA v1, p.2500, 2012. ,
Microinjection and Live-cell Imaging Zygotes were injected with 5 ng/ml mRNAs encoding EGFP-a-tubulin and H2B-mCherry at 5 hours post fertilization, Raw images were stacked and projected by CV1000 Software, 2009. ,
QUANTIFICATION AND STATISTICAL ANALYSIS Reference Sequences RefSeq transcript coordinates (RefFlat) and assemblies (GTF) of mouse genome mm10 were obtained from iGenomes (Illumina), The repeat masker track of mm10 was downloaded from UCSC Table Browser, 2011. ,
Windows with less than 5 informative CG (or non-CG) sites were excluded. To study CG methylation at ERVs, reads were mapped to the Repbase annotations. For allelic-specific analysis, reads were mapped to N-masked mm10 genome using published SNP data of JF1, WGBS Data Analysis Reads were trimmed to remove low quality bases and adaptor sequences using Trim-Galore! v0, vol.3, 2011. ,
ERVs with R 100 copies in the genome and at least 2-fold increase in all replicates were defined as derepressed. RefSeq transcripts containing an extra ERV sequence at their 5 0 ends were identified as additional chimeric transcripts. For allelic-specific analysis, reads were mapped to N-masked mm10 genome using published SNP data of JF1, RNA-seq Data Analysis Reads were trimmed and mapped to mouse genome mm10 by HISAT2, vol.1, 2012. ,