title: GSM8705164: N2A, ADA-Clone1, HS18, rep1, 4C-seq; Mus musculus; OTHER
library_construction_protocol: Briefly, cortical tissues were dissected out using fine forceps and immediately transferred to a dish containing ice-cold NeuroBasal medium. The tissues were then torn into pieces using fine forceps and transferred to an Eppendorf tube containing 0.25% trypsin-EDTA. The sample was incubated at 37°C for 15 min with continuous shaking at 300 rpm. The trypsin digestion of tissues was quenched by the addition of 10% FBS in the PBS buffer. After filtering the dissociated tissues into a single-cell suspension, the volume was adjusted to 5 ml using 10% FBS in the PBS for crosslinking. Quantitative high-resolution chromosome conformation capture copy (QHR-4C) experiments were performed as previously described with minor modifications. In brief, five million cells were harvested as described above. The cells were resuspended in MEM medium, and then crosslinked with 2% formaldehyde at room temperature with slow rotations for 10 min. The reaction was quenched by the addition of 2 M glycine. The fixed cells were spun down at 900 × g for 5 min at 4°C and then washed twice with 1 ml of ice-cold PBS. Cells were lysed twice with 1 ml of ice-cold 4C permeabilization buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.2% SDS, 0.5% NP40, 1% Triton X-100, and 1 × protease inhibitors) with slow rotations for 10 min at 4°C. Cells were then resuspended in nuclease-free water, followed by the addition of 10 × DpnII buffer and 10% SDS, and incubation at 37°C with constant shaking at 900 rpm. After 1 h, 20% Triton X-100 was added to the sample, and the sample was incubated at 37°C for an additional hour with constant shaking at 900 rpm. Next, DpnII was added to the sample, followed by an overnight incubation at 37°C with constant shaking at 900 rpm. The enzyme was heat-inactivated at 65°C for 20 min. The sample was centrifuged at 2,000 × g for 10 min, and the supernatant was carefully removed to not disturb the cell pellets. The cell pellets were then resuspended with nuclease-free water. The digested nuclei were ligated with T4 DNA ligase in a total ligation volume of 2 ml at 16°C for 12 h. The next day, the ligated chromatin was digested with 10 mg/ml Protease K at 65°C for 2 h. The DNA was extracted using phenol-chloroform extraction, followed by ethanol precipitation as described above. The DNA pellet was then dissolved in nuclease-free water and sonicated using a Bioruptor system with a low energy setting. Sonication was performed in cycles of 30-sec on and 30-sec off for 12 cycles to obtain DNA fragments ranging from 200-600 bp. The concentration of the sonicated DNA was measured using the Qubit fluorescent dye. Next, DNA was used as a template for linear amplification with a specific 5' biotin-tagged primer complementary to the viewpoint. The linear amplification was conducted in a total volume of 0.05 ml, including 2 × Buffer, dNTP, Polymerase, and 1 uM 5' biotin-tagged primer, using the program (95°C, 2 min; 95°C, 15 s, 58°C, 25 s, 72°C, 1 min 30 s for 80 cycles; and a final extension at 72°C, 5 min). The amplified products were denatured at 95°C for 5 min and then chilled on ice to obtain biotinylated single-stranded DNA (ssDNA). The biotinylated ssDNA was ligated with 50 mM Adapter-4C adapters for 12 h at 16°C and purified by streptavidin magnetic C1 beads. The QHR-4C libraries were generated via PCR amplification (94°C, 2 min; 94°C, 10 s, 60°C, 15 s, 72°C, 1 min for 19 cycles; and a final extension at 72°C, 5 min) using high-fidelity DNA polymerase. The amplified PCR products were purified using the High-Pure PCR Product Purification kit and sequenced on an Illumina NovaSeq 6000 platform.