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We collected tissue that was cross-linked for 10 minutes with 1% final concentration fresh formaldehyde and quenched with 0.2 M final concentration glycine for 5 minutes. The cross-linked cells were subsequently lysed in lysis buffer (10 mM Tris-HCl (pH 8.0), 10 mM NaCl, 0.2% NP40, and complete protease inhibitors [Roche]). The extracted nuclei were resuspended with 150 uL 0.1% sodium dodecyl sulfate (SDS) and incubated at 65 C for 10 minutes. Then SDS molecules were quenched by adding 120 uL water and 30 uL 10% Triton X-100 and incubated at 37 C for 15 minutes. The DNA in the nuclei was digested by adding 30 uL 10x New England Biolabs (NEB) buffer 3.1 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 100 ug/mL bovine serum albumin (BSA), pH 7.9) and 150 U of DPN II and incubated at 37 C overnight. On the next day, the DPN II enzyme was inactivated at 65 C for 20 minutes. Next, the cohesive ends were filled in by adding 1 uL 10 mM dTTP, 1 uL 10 mM dCTP, 1 uL 10 mM dGTP, 2 uL 5 mM Biotin-14-dATP, 14 uL water, and 4 uL (40 U) Klenow and incubated at 37 C for 2 hours. Subsequently, 663 uL water, 120 uL 10x blunt-end ligation buffer (300 mM Tris-HCl, 100 mM MgCl2, 100 mM DTT, 1 mM ATP, pH 7.8), 100 uL 10% Triton X-100, and 20 U T4 DNA ligase were added to start proximity ligation. The ligation reaction was held at 16 C for 4 hours. After ligation, the cross-linking was reversed with 200 ug/mL proteinase K (Thermo) at 65 C overnight. DNA purification was achieved using QIAamp DNA Mini Kits (Qiagen) according to the manufacturer's instructions. Purified DNA was sheared to a length of 400 bp. |
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