GSM5550218: EPS-shYy1-H3K27ac_cut_tag; Mus musculus; OTHER
For RNA-seq, Total RNAs were extracted with TRIzol. For quantitative PCR, cDNAs were synthesized with ReverTra Ace (Toyobo) and oligo dT (Takara), and then analyzed by qPCR with SYBR green mixture (Genstar) and performed on a CFX Real-Time System (Bio-Rad). For 10 × Genomics single cell RNaseq, the cells cultured in F4H medium for 6 days were dissociated with 0.25% TrypLE Express Enzyme at 37°C for 10 min. Single cell populations were resuspended in PBS with 0.04% BSA. Single cell libraries were created using Chromium Single Cell 30 Reagent Kits and sequenced on a Novaseq 6000 sequencer. Approximately 10000 cells were captured for each time point.CUT&Tag experiments were performed as previously described65. In brief, 100,000 cells were harvested washed twice with 200 µL Wash Buffer. 10 µL Concanavalin A beads (Bangs Laboratories) were added per sample and incubated at room time for 15 min. Add 100 µL Dig-wash Buffer containing 2 mM EDTA and 1µg primary antibody. Primary antibody incubation was performed on a rotating platform overnight at 4 °C. Add 200 µL Dig-Wash buffer to remove unbound antibodies. Then incubated with pAG-Tn5 (home-made) at 4 °C for 2 h. Add 200 µL Dig-med Buffer to remove unbound pAG-Tn5 protein. Next, cells were resuspended in 100 µL tagmentation buffer and incubated at 37 °C for 1 h. To stop tagmentation, 2.25 µL of 0.5 M EDTA, 2.75 µL of 10% SDS and 0.5 µL of 20 mg/mL Proteinase K was added to 50 µL of sample, which was incubated at 55 °C for 30 min, and then at 70 °C for 30 min to inactivate Proteinase K. Then extract the DNA. Antibodies included: H3K27ac (Active Motif, 39133). RNA sequencing libraries were constructed using the VAHTS mRNA-seq V3 Library Prep Kit for Illumina (Vazyme).