GSM8187100: GABPA_CUTrun_E35_ICM_rep1; Mus musculus; OTHER
For mESC CUT&RUN with more than 10K, cells were resuspended in 50 μl wash buffer (20 mM HEPES/pH=7.5, 150 mM NaCl, 0.5 mM spermidine and 1× protease inhibitor) with activated Concanavalin A Magnetic Beads (Polysciences, 86057-3) for 10 mins at RT, then samples were included with anti-GABPA (1:40, Proteintech, 21542-1-AP, lot#00018047) overnight at 4°C. Samples were included with 2.8 ng/μl pA-MNase (home-made) for 2 hrs at 4 °C. Subsequently, samples were included with 200 μl pre-cooled 0.5 μM CaCl2 for 20 mins at 4 °C and quench by adding 23 μl 10×stop buffer (1700 mM NaCl, 20 mM EGTA, 100 mM EDTA , 0.02% Digitonin, 250 µg/ml glycogen and 250 µg/ml RNase A). DNA Fragments were released by incubation at 37°C for 15 mins. 2.5 μl 10% SDS and 2.5 μl 20 mg/ml Protease K (Thermo Fisher) was added and incubated at 55 °C for at least 1 h. DNA was extracted by phenol-chloroform followed by ethanol precipitation. For low input CUT&RUN, mESC, zona-free embryos or isolated ICM were resuspended in 50 μl wash buffer with activated Concanavalin A Magnetic Beads for 10 mins at RT, then samples were fixed with 1% formaldehyde for 1 min. After three times wash with wash buffer, samples were included with anti-GABPA overnight at 4°C. The followed protocol is same as previous one. Sequencing libraries were prepared with NEBNext Ultra II DNA library preparation kit for Illumina (New England Biolabs, E7645S).  For ATAC-seq, ESC and isolated ICM were digested with adapter-loaded Tn5 for 15 mins at 37°C, and stopped by stop buffer (100 mM Tris/pH=8.0, 100 mM NaCl, 40 µg/ml Proteinase K and 0.4% SDS) and incubated overnight at 55°C. 5 µl of 25% Tween-20 was added to quench SDS. Sequencing libraries were prepared with NEBNext High-Fidelity 2×PCR Master Mix (NEB, M0541S).  For RNA-seq, fresh ESC or embryos were collected. Reverse transcription and cDNA amplification were performed with SMART-Seq™ v4 Ultra™ Low Input RNA Kit (Clontech, 634890), followed cDNA fragmentation, adaptor ligation, and amplification using Nextera® XT DNA Sample Preparation Kit kit (Illumina, FC-131-1024).