GSM9181545: H9 embryonic stem cell replication2; Homo sapiens; OTHER
Fresh cell cultures were harvested and counted to obtain 1×10^⁵ cells per sample.  Cells were then incubated with Concanavalin A-coated beads at room temperature for 10 min.  After removal of the supernatant, cells were resuspended in ice-cold Antibody Buffer containing the PAX6 antibody (Cell Signaling Technology, #60433) and incubated overnight at 4˚C.  The primary antibody solution was then removed, and the cells were incubated with the secondary Goat Anti-Rabbit IgG H&L (Vazyme, Ab206-01) diluted in DIG Wash Buffer for 1 h at room temperature.  After thorough washing, CUT&Tag pA/G-Tn5 Transposomes were added and incubated for 1 h at room temperature.  Following additional washes, tagmentation was performed at 37˚C for 1 h.  DNA was extracted using magnetic DNA purification beads. The purified DNA was amplified via PCR using indexed i7 and i5 primers (Vazyme, TruePrep DNA Library Prep Kit V2 for Illumina, #TD501).