title: GSM8685121: HiC_E14.5Ngn3flGFPH; Mus musculus; Hi-C
library_name: GSM8685121
library_construction_protocol: Hi-C was performed with modifications to the DNA fragmentation and library construction steps. Briefly, E14.5 Ngn3high cells were fixed with 1% formaldehyde at room temperature (RT) for 10 minutes, and the crosslinking reaction was quenched with glycine for 10 minutes at RT. Cells were washed with 1 × PBS and lysed in 50 μl lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 0.1 mM EDTA, 0.5% NP-40, and protease inhibitors) on ice for 50 minutes. After centrifugation at 3,000 r.p.m. at 4 °C for 5 minutes, the supernatant was carefully discarded. Chromatin was solubilized in 10 μl 0.5% SDS and incubated at 62 °C for 10 minutes, followed by quenching with 5 μl 10% Triton X-100 at 37 °C for 30 minutes. The nuclei were digested with 50 U MboI at 37 °C overnight with rotation in a final volume of 50 μl. MboI was inactivated at 62 °C for 20 minutes. For biotin labeling, 1.5 μl 1 mM dATP, 1.5 μl 1 mM dGTP, 1.5 μl 1 mM dTTP, 3.75 μl 0.4 mM biotin-14-dCTP, and 10 U Klenow enzyme were added, and the reaction was incubated at 37 °C for 1.5 hours with rotation.        Ligation was performed by adding 60 μl ligation mix (38.8 μl water, 12 μl 10 × NEB T4 DNA ligase buffer, 7 μl 10% Triton X-100, 1.2 μl 10 mg/ml BSA, and 1 μl 400 U/μl T4 DNA ligase) and incubating at RT for 6 hours with rotation. Crosslinking was reversed, and the DNA was purified. DNA was sheared to 300–500 bp using a Q800R3 sonicator. Biotin-labeled DNA fragments were captured with 10 μl Dynabeads MyOne Streptavidin C1 (Life Technologies). The libraries were subjected to 150 bp paired-end sequencing on the Illumina NovaSeq 6000