ZBTB5 CUT&RUN-Seq in 293T cell line
For library construction, ZBTB5 3Flag knock-in 293T cells were harvested and quantified, according to the manufacturers protocol for the CUT&RUN Assay Kit (Vazyme, Nanjing, China). The cells were incubated with Concanavalin A-coated magnetic beads (ConA Beads) at room temperature for 10 minutes. Each sample was resuspended in 100 L of pre-chilled Antibody Buffer and incubated with an anti-Flag antibody. The cell-bead complexes were then rotated at 4 C for 12 hours. Following antibody binding, the beads were collected and incubated with pG-MNase enzyme at 4 C for 1 hour with rotation. DNA fragmentation was initiated by adding CaCl2 in Dig-wash Buffer under a combination of ice and room temperature incubations. The reaction was terminated with Stop Buffer to release DNA fragments. Purification of the fragmented DNA was carried out using a spin column-based method.  The resulting libraries were quantified with the Qubit 3.0 Fluorometer (Thermo Scientific, MA, USA). Fragment size distribution was evaluated using the Agilent 4200 system (Agilent Technologies, CA, USA). Library molar concentration was measured by quantitative PCR (qPCR) on the QuantStudio 5 Real-Time PCR System (Thermo Scientific, MA, USA). Finally, sequencing was conducted on the Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA) using the NovaSeq 6000 S4 Reagent Kit (v1.5, 300 cycles; catalog number: 20028312) in accordance with the manufacturers instructions.
SUB15563903
Sample01