After cross-link reversal, 1μl of RNase A was added to each sample, followed by incubation at 37 ℃ for 30 min. DNA was purified using phenol:chloroform:isoamyl alcohol (25:24:1, v/v/v), followed by ethanol precipitation. The pellet was resuspended in 90 μl of TLE buffer (10 mM Tris-HCl pH 8.0, 0.1mM EDTA). Biotin removal was performed in a 100μl reaction containing 1x NEBuffer 2.1, 25μM each of dATP and dGTP, and 30 units of T5 DNA polymerase. The reaction was incubated at 20 ℃ for 4 h, then heat-inactivated at 75 ℃ for 20 min. DNA was fragmented using a Covaris S220 sonicator to an average fragment size of ~400 bp (peak power: 140W; duty factor: 10%; cycles per burst: 200; duration: 55 s). Fragment size selection was performed using a dual-sided AMPure XP bead cleanup: 0.6x bead volume for initial selection and 0.25x for secondary purification. Biotinylated fragments were captured using 10μl of MyOne C1 streptavidin beads per sample. The beads were washed with Tween wash buffer (5 mM Tris-HCl pH 8.0, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween-20) and resuspended in 400μl of 2x binding buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 2 M NaCl). DNA diluted in TLE (400μl) was added to beads and incubated for 15 min at RT on a nutator. The beads were sequentially washed with 400μl of 1x binding buffer and 100 μl of 0.1x TE buffer (10mM Tris-HCl pH 8.0, 1mM EDTA), resuspended in 50μl of 0.1x TE, and transferred to 0.2 ml PCR tubes. Library preparation was conducted using the NEBNext Ultra II DNA library Prep Kit (NEB, #E7645L), with slight modifications to the standard protocol. Following adaptor ligation, the beads were transferred to DNA LoBind tubes and washed twice with 400μl of Tween wash buffer for 5 min at RT on a nutator, then with 200μl of 1x binding buffer and 200μl of 0.1x TE. Final elution was performed in 15μl of 0.1x TE by heating at 98 ℃ for 10 min. Supernatants were collected and subjected to PCR amplification for 10 cycles. Amplified libraries were purified using 0.9x AMPure XP beads and sequenced on an Illumina Novaseq X platform (150bp paired-end reads).