title: GSM9559626: SFN_micro_c_S074_JC4; Mus musculus; MNase-Seq
library_construction_protocol: MNase digestion For each cell line, 100,000 cells were collected and cross-linked with 1% formaldehyde at room temperature. The reaction was quenched with glycine at a final concentration of 1 M, followed by one wash with PBS. The cells were then subjected to a second cross-linking step using 3 mM EGS crosslinker (MCE, HY-130458), which was again quenched with 1 M glycine. After a final PBS wash, the cells were lysed on ice with cell lysis buffer (10 mM Tris pH 8.0, 10 mM NaCl, 0.2% NP-40) for 20 min and subsequently digested with MNase (Micrococcal Nuclease, NEB，M0247S) at a 1:500 dilutions in 30 min. T4 ligase ligation After chromatin fragmentation by MNase, a master mix was prepared containing T4 ligase buffer, biotin-dATP (Active-Motif, 14139), biotin-dCTP (MCE, HY-D1668), dGTP, dTTP, 100 mM ATP (Thermo, R1441), T4 PNK (T4 polynucleotide kinase, NEB, M0201L), Klenow fragment (NEB, M0210L), T4 DNA ligase (Thermo, EL0013), BSA, and EGTA. Cell pellets were resuspended in this mix and incubated first at 25 ℃ for 2 hours, then at 37 ℃ for 2 hours, followed by a final incubation at 16 ℃ for at least 8 hours to ensure efficient ligation. Following proximity ligation, unligated DNA ends were digested with Exonuclease III (NEB, M0206L) at 37℃ to reduce background noise in subsequent sequencing. Reverse Cross-linking and DNA Extraction The sample was normalized to a final volume of 100 μL with ddH₂O. The reaction was supplemented with 12.5 μL of Proteinase K (20 mg/mL), 12.5 μL of 10% SDS, and 1 μL of RNase A (10 mg/mL), yielding final concentrations of 2.5 mg/mL, 1.25%, and 0.1 mg/mL, respectively. Following incubation at 37℃ for 30 min, the samples were heated at 65℃ for at least 3 h to reverse cross-links. DNA was then purified using 0.8 volumes of DNA purification beads according to the manufacturer's instructions and eluted for library construction. Biotin Pull-Down 15 μL of Dynabeads Myone Streptavidin C1 beads (Thermo, 65002) were placed on a magnetic stand and washed three times with BW buffer (Binding and Washing buffer, 5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl). The beads were then resuspended in 50 μL of 2x BW buffer, followed by the addition of 50 μL of purified DNA. The mixture was incubated at room temperature for 25 minutes. After incubation, the beads were washed once with BW buffer and twice with 1xT4 ligase buffer. Finally, the beads were resuspended in 50 μL of DNase free water for subsequent library construction. DNA Library Construction A commercial DNA library construction kit (VAHTS Universal DNA Library Prep Kit for Illumina V4 (ND610)) was used for library preparation. The main procedure consisted of four stages: DNA end repair, adapter ligation, library amplification, and library purification. Following adapter ligation, beads were washed three times with BW buffer to reduce adapter-related background. Library amplification was typically performed for 9-10 cycles. Finally, the DNA library was purified using 0.9x DNA clean beads. To adapt to the MGI DNBSEQ‑T7 sequencing platform, all adapters and amplification primers were replaced with their MGI-compatible counterparts. Micro-C, DNA libraries were prepared on-bead using the VAHTS Universal DNA Library Prep Kit (Vazyme), including end repair, dA-tailing, and adapter ligation. Libraries were PCR-amplified (9–11 cycles), size-selected with VAHTS DNA Clean Beads, and sequenced on Illumina NextSeq500 or MGI DNBSEQ-T7. Basecalling was performed using platform-specific software (NextSeq Control Software v2.2.0 or Basecall_T7_GPU_1.2.0.26_Centos).