title: GSM8213824: H3K27me2 morula rep2; Mus musculus; ChIP-Seq
library_construction_protocol: For superovulation, 4- to 10-week-old B6N females were injected with 7.5 IU pregnant mare serum gonadotropin (ASKA Pharmaceutical) or 0.15 ml CARD HyperOva (Kyudo Co., Ltd) followed by 7.5 IU human chorionic gonadotropin (hCG) (ASKA Pharmaceutical) at a 48-h interval. Cumulus-oocyte complexes (COCs) were harvested 15–17 h after human chorionic gonadotropin injection. For in vitro fertilization, COCs were inseminated with activated spermatozoa in Human Tubal Fluid medium supplemented with 10 mg/ml BSA (Merck Millipore; 12657). Spermatozoa were obtained from the caudal epididymis of adult PWK/PhJ mice [RBRC00213 (RIKEN BioResource Research Center) originated from 003715 (The Jackson Laboratory)] and B6D2F1 (BDF1) males (Jackson Laboratory). Spermatozoa were capacitated by 1 h incubation in CARD FERTIUP (Kyudo) for PWK sperm or in Human Tubal Fluid medium for BDF1 sperm. At 6–8 h postinsemination, zygotes with two pronuclei were transferred to KSOM and cultured under a humidified atmosphere with 5% CO2/95% air at 37.8°C. For CATCH-seq and CUT&RUN sample collection, polar bodies were removed as described previously (Hayashi and Inoue 2023). Four-cell, eight-cell, morula and blastocyst embryos, were collected at 46–52, 60, 72 and 120 h post-insemination with 500-1,000 blastomeres for each CATCH-seq experiment. CATCH-seq is an improved method of the ultra low-input native ChIP-seq (ULI-NChIP) (Brind'Amour et al. 2015), and the original protocol of CATCH-seq was described in (Zhu et al. 2021). We have made some changes in CATCH-seq to improve the usability. Cells were collected to 9 µL of Nuclei EZ lysis buffer (Sigma, NUC-101) supplemented with complete EDTA-free protease inhibitor cocktail and 1 mM phenylmethanesulfonyl fluoride. The samples were snap frozen in liquid nitrogen and stored at -80°C  until use. At the day of library construction, the samples were thawed on ice, and added with 1 µL of the lysis buffer containing a constant number of Drosophila melanogaster S2 cells (Thermo Fisher Scientific, R69007) for spike-in normalization purpose. Then, 1 µL of 1% Triton X-100 (Merck 93443) and 1% deoxycholate (Nacalai, 10712-54) mixture solution was added to the samples, and sit on ice for 5 min. The chromatin was fragmented by 2 U/µl MNase (M0247S, NEB) in 1xMNase buffer supplemented with 1% PEG6000 (HAMPTON RESEARCH, HR2-533) and 2 mM DTT (Nacalai) at 21 or 37°C  for 7.5 min. The digestion temperature was dependent on the experimental condition, including the lot of MNase and the freshness of PEG6000, which had been determined by fragment sizes of input libraries in a preliminary test. The MNase reaction was stopped by adding 1/10 volume of 100 mM EDTA and 1/12 volume of the 1% Triton X-100 and 1% deoxycholate mixture, and the samples were rest on ice for 15 min. The chromatin lysates were then added with freshly prepared immunoprecipitation buffer (Brind'Amour et al. 2015), and 5% volume was kept for input library construction. To the rest of the lysate, 30 ng of annealed I-SceI carrier DNA was added (Zhu et al. 2021). The forward and reverse strands of the carrier DNA were as follows: /5AmMC6/Gtagggataacagggtaattagggataacagggtaattagggataacagggtaattagggataacagggtaattagggataacagggtaattagggat aacagggtaat*c/3AmMO/ and /5AmMC6/Gattaccctgttatccctaattaccctgttatccctaattaccctgttatccctaattaccctgttatccctaattaccctgttatccctaattaccctgttatcc cta*c/3AmMO/, respectively, where asterisks represent phosphorothioate bonds. The oligos were synthesized by Integrated DNA Technologies. For each immunoprecipitation reaction, 0.5 µL of rabbit anti-H3K27me2 (Cell Signaling Technologies, 9728), 500 ng of rabbit anti-H3K27me3 (Millipore 07449), or 250 ng of rabbit anti-H3K27ac (Diagenode C15410196) conjugated to precleared Dynabeads Protein A (Thermo Fisher Scientific, 10006D) and G (Thermo Fisher Scientific, 10007D) mixture was used. The quality of these antibodies was validated in previous studies [H3K27me2 (Ferrari et al. 2014a; Juan et al. 2016; Hojfeldt et al. 2018b; Lavarone et al. 2019; Meng et al. 2020), H3K27me3 (Adli et al. 2010; Kelly et al. 2019; Ren et al. 2019), H3K27ac (Zenk et al. 2021)]. After immunoprecipitation at 4°C overnight, the chromatin-Dynabeads were washed twice each by the low and high salt wash buffers, and the chromatin was eluted in the freshly prepared ChIP elution buffer at 65°C for 1 hr (Brind'Amour et al. 2015). DNA was recovered by phenol-chloroform extraction followed by ethanol precipitation. Adaptor ligation was performed by NEBNext Ultra II DNA Library Prep Kit for Illumina (E7645, NEB) in a half scale of the manufacturer's instruction, and the libraries were purified by 1.8x SPRIselect beads (B23318, Beckman Coulter). The DNA was amplified by KAPA Hifi 2X mater PCR mix (KK2605) for 13-15 PCR cycles with dual indexing primers (NEBNext Multiplex Oligos for Illumina, E6440). After purification with 0.9x SPRIselect beads, the samples were digested by I-SceI (5 U/µl, NEB, R0694) 37°C for 2 hrs followed by heat inactivation at 65°C for 20 min and purified by 0.9x SPRIselect beads. The second amplification was not performed. The libraries were sequenced on Nextseq500 (single-end reads) or Nextseq2000 (paired-end reads) (Illumina). CUT&RUN libraries were prepared as previously described (Hayashi and Inoue 2023). For each CUT&RUN library, 100-180 morula embryos were used per replicate, and the same number of embryos were used to compare between control and mutant embryos. The antibodies used in this study were rabbit anti-JARID2 (1/50; Cell Signaling Technologies, 13594), rabbit anti-SUZ12 (1/50; Cell Signaling Technologies, 3737), and rabbit anti-H3K36me3 (1/500; Abcam 9050) antibodies. The specificity of these antibodies was proven in previous studies (Hojfeldt et al. 2018; Healy et al. 2019; Hojfeldt et al. 2019; Glancy et al. 2023). Protein A-MNase was provided by S. Henikoff 's laboratory (Skene and Henikoff 2017). For quantitative CUT&RUN, 20 pg of yeast DNA was added to each sample. CUT&RUN libraries were sequenced on a NextSeq 500 with single-end reads (Illumina).