GSM8386636: EdUseq_mESC_MSO_Ai_4h; Mus musculus; OTHER
Round cells were lifted by tapping the sides of the flasks and visually inspected. Depending on the conditions of the experiment, cell suspensions from different flasks were strained through a 40 µm mesh and pooled into 50 mL tubes, centrifuged at room temperature (400G, 5 minutes), washed once with 15 mL of PBS and centrifuged again. Pelleted cells were then carefully resuspended and plated. Commonly, the cells lifted from two T225 flasks would be seeded on one 10 cm plates (for EdU-seq and EU-seq) or on one 6 or 12 multi-well plate (if used for cell cycle analysis). Thymidine or aphidicolin synchronisation. Pluripotent cells (0.5-1x106 mESCs, ZHBTc4 or 2TS22c) or MEFs (1.5-2x106 cells) were seeded on 10 cm plates. 24 to 48 hours later 2mM thymidine or 4µM aphidicolin was added to the media for 10 to 12 hours. Cells were washed three to four times with PBS and released into fresh media. MEFs were also synchronised by 60 hours of serum starvation (DMEM, 0.1% FBS, P/S, L-glutamine) and released into fresh media. Purified EdU-labeled DNA was used for library preparation with TrueSeq ChIP Library Preparation Kit (Illumina) and high-throughput 100-bp single-end sequencing was performed on Illumina NovaSeq 6000 sequencer.