GSM9465091: HRO_antiKDM6A_siluc_rep1; Homo sapiens; OTHER
CUT&TAG sample preparation was performed following protocols from the Fred Hutchinson Cancer Research Center. Approximately 50,000 harvested and counted cells were resuspended in Wash Buffer and mixed with Concanavalin A-coated magnetic beads. The mixture was incubated at room temperature to allow binding. Bead-bound cells were then washed and incubated with a primary antibody(anti-KDM6A, invitrogen, PA5-31828, 1μg) specific to the target protein, followed by a secondary antibody for bridging. Next, the bead-bound cells were incubated with Protein A-Tn5 transposase in the presence of digitonin for tethering. After a brief incubation to allow the transposome to tether to chromatin sites targeted by antibodies, cells were washed and subjected to magnesium activation to induce tagmentation. This step facilitated DNA cleavage and simultaneous tagging with sequencing adapters. Following tagmentation, DNA was purified using SPRI beads and quantified using a fluorometric method. Libraries were PCR-amplified with barcoded primers, cleaned up, and subjected to quality control checks, including size selection and quantification.