GSM9536672: CUT&Tag hek293t_emx1_h3k9me3_rep1; Homo sapiens; OTHER
CUT&Tag libraries were generated using the Hyperactive Universal CUT&Tag Assay Kit for Illumina (Vazyme, TD903) following the manufacturer's instructions with minor adjustments. Harvested cells were washed once with 500 μL Wash Buffer and resuspended in 100 μL Wash Buffer. Ten microliters of Concanavalin A beads were added and incubated at room temperature for 10 minutes with intermittent gentle inversion. Bead-bound cells were resuspended in 50 μL pre-chilled Antibody Buffer containing primary antibody against CTCF (Active Motif, 61932), H3K27ac (Abcam, ab4729), H3K9me3 (Active Motif, 39161), or H3K27me3 (Cell Signaling, C36B11), and incubated overnight at 2-8°C. On day 2, beads were washed and resuspended in 50 μL Dig-wash Buffer supplemented with 0.01 mg/mL secondary antibody (Novoprotein, N269), incubated at room temperature for 30-60 minutes on a tube rotator, and washed three times with 200 μL Dig-wash Buffer. Beads were resuspended in 100 μL Dig-300 Buffer containing 2 μL pA/G-Tnp transposome and incubated for 1 hour at room temperature with gentle rotation. After three washes with 200 μL Dig-300 Buffer, beads were resuspended in 50 μL tagmentation buffer (5× TTBL diluted in Dig-300 Buffer). Tagmentation was performed at 37°C for 60 minutes in a thermocycler with heated lid disabled. To stop reaction and extract DNA, 5 μL Proteinase K, 100 μL Buffer L/B, and 20 μL DNA Extract Beads were added, and DNA was eluted in 20 μL nuclease-free water. Purified DNA was amplified using CUT&Tag Amplification Mix (CAM) and indexed primers (12-14 cycles for ~10,000 cells), then final PCR products were purified using VAHTS DNA Clean Beads (Vazyme, N411). Sequencing libraries were generated on the Illumina NovaSeq 6000 platform.