GSM9316920: H3K4me3_P1P6MLL_WT_blastocyst_Rep1; Mus musculus; ChIP-Seq
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). CATCH-seq is an improved method of the ultra-low-input native ChIP-seq (ULI-NChIP). The original protocol of CATCH-seq was described in Zhu et al., and we have made some modifications to improve its usability. For each immunoprecipitation reaction, 0.25 µL of rabbit anti-H3K4me3 (Active Motif 39159, 70 oocytes/library), 500 ng of rabbit anti-H3K27me3 (Diagenode C15410069, 50-80 oocytes/library), 1.0 µg of mouse anti-H3K36me2 (MBL, MABI0332, 100 oocytes/library) or 1.0 µL of rabbit anti-H3K36me3 antibody (Active Motif 61022, MABI0333, 100 oocytes/library) conjugated to precleared Dynabeads Protein A (Thermo Fisher Scientific, 10006D) and G (Thermo Fisher Scientific, 10007D) mixture was used. CUT&RUN libraries were prepared as previously described (Hayashi, R. & Inoue, A). The antibodies used were rabbit anti-H3K27me3 (1/100, Diagenode C15410069, 150-170 oocytes/library). Protein A-MNase was provided by Dr. Steven Henikoff's laboratory (Skene et al.). CUT&RUN libraries were sequenced on a NextSeq500 or Nextseq2000 with paired-end or single-end reads (Illumina).