title: GSM8656518: H3K36me2 H2A.Z-CTR FGO rep2; Mus musculus; OTHER
library_construction_protocol: After ZP of SN-type FGOs were removed by acid tyrodes' solution, ZP-free oocytes 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 -80C 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 21C or 37C 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). CUT&RUN libraries were prepared as previously described (Hayashi and Inoue 2023). The antibodies used were mouse anti-H2A (1/100, MBL D210-3), rabbit anti-H2A.X (1/100, Abcam 11175), and rabbit anti-TH2A antibody (1/100) (Shinagawa et al. 2014). Protein A-MNase was provided by the Steven Henikoff Lab (Skene and Henikoff 2017). For CATCH-seq, 3 uL of rabbit anti-H2A.Z (Active motif 39113, 200 oocytes/library), 500 ng of mouse anti-H2A (MBL D210-3, 200 oocytes/library), 1 uL of rabbit anti-H2A.X (Abcam ab11175, 200 oocytes/library), 500 ng of rabbit anti-TH2A antibody (a gift from Shunsuke Ishii, 200 oocytes/library) (Shinagawa et al. 2014), 0.25 uL of rabbit anti-H3K4me3 (Active Motif 39159, 40 oocytes/library), 500 ng of rabbit anti-H3K27me3 (Diagenode C15410069, 80 oocytes/library), or 1.0 µl of rabbit anti-H2AK119ub1 antibody (Cell Signaling Technology 8240, 500 oocytes/library) 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 [H2A.Z (Murphy et al. 2018), H2A (Inoue and Zhang 2014), H2A.X (Inoue and Zhang 2014), TH2A (Shinagawa et al. 2014), H3K4me3 (Kaya-Okur et al. 2020), H3K27me3 (Inoue et al. 2018), H2AK119ub1 (Mei et al. 2021)]. After immunoprecipitation at 4C 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 65C 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) 37C for 2 hrs followed by heat inactivation at 65C for 20 min and purified by 0.9x SPRIselect beads.