GSM9000817: KO_L2C_ULI-MNase-seq_r1; Mus musculus; MNase-Seq
20-30 blastomere per replicate were isolated and washed before they were placed into 1.4 μL of lysis buffer (10 mM Tris-HCl, pH 8.5, 5 mM MgCl2, 0.6% NP-40) for individual reactions. Then, 1.8 μL of MNase master mix (MNase buffer, 0.125 U/μL MNase (NEB, M0247S), 2 mM DTT, and 5% PEG 6000) was added into each tube, and the reaction was incubated at 25 °C for 10 min for chromatin fragmentation. The reaction was stopped by the addition of 0.4 μL of 100 mM EDTA, and then 0.4 μL of 2% Triton X-100 was added to the reaction to release the fragmented chromatin. Then, 0.2 μL of 20 mg/mL protease (Qiagen) was added, and the reaction was incubated at 50 °C for 90 min for protein digestion followed by incubation at 75 °C for 30 min for protease inactivation. The sequencing libraries were prepared using the KAPA Hyper Prep kit for the Illumina platform (Roche, kk8504) following the manufacturer's instructions. After standard procedures including end repair and A-tailing, adapter ligation, post-ligation cleanup, and library amplification, the resulting products were subjected to a second round of PCR amplification with the same provided primers to generate sufficient DNA materials for highthroughput sequencing. For mouse embryos, the sequencing libraries were prepared using KAPA Stranded RNA-Seq Kit with RiboErase(Kapa Biosystems, KK8483) and KAPA HyperPrep Kit for the Illumina platform (Roche, kk8504) following manufacturer's protocol. Paired-end sequencing with 150-bp read length was performed on the HiSeq X Ten (Illumina) platform.