Method for Simultaneously Enriching Intrinsically Disordered Proteins and Associated Three-Dimensional Genomic Structures Thereof

A method for simultaneously enriching intrinsically disordered proteins (IDPs) and associated three-dimensional genomic structures thereof, comprising the steps of:

• cross-linking and lysing test cells, and extracting nuclei;
• subjecting the extracted nuclei to enzymatic digestion with restriction endonuclease Dpn II, end repair, and proximity ligation;
• subjecting the proximity-ligated nuclei to sonication to obtain a sonicated product;
• dividing the sonicated product into two parts to obtain product I and product II;
• enriching sonicated DNA-protein complexes from the product I with a biotinylated isoxazole, and subjecting the sonicated DNA-protein complexes to washing, elution, and protease treatment;
• extracting and purifying DNA after the protease treatment, and constructing a sequencing library from the purified DNA;
• and treating the product II with a nuclease, extracting proteins, and performing protein mass spectrometry analysis;wherein the test cells are cross-linked using formaldehyde and disuccinimidyl glutarate;
• wherein the cross-linking comprises the steps of:
• resuspending the test cells in 1×phosphate-buffered saline (PBS) solution, adding formaldehyde for incubation for 8-15 min, then terminating a reaction by adding glycine;
• after terminating the reaction, washing resulting cells with 1×PBS solution, and resuspending the washed cells in 1×PBS solution containing 3 mM disuccinimidyl glutarate, incubating re resuspended cells at room temperature for 30-50 min, then adding glycine for continued incubation;
• washing the incubated cells with 1×PBS solution containing 0.05% bovine serum albumin, adjusting cell concentration, performing centrifugation, discarding a resulting supernatant, and collecting a resulting cell pellet for cryopreservation in liquid nitrogen;
• wherein the enzymatic digestion comprises the steps of adding and mixing 10-15 μL 10× NEBuffer 3.1 and 400 U restriction endonuclease Dpn II following cell lysis, digesting a resulting mixture on a 37 °C shaker overnight, followed by incubation at 60-68 °C for 15-25 min to inactivate the restriction endonuclease Dpn II, cooling on ice, and performing the end repair, and the proximity ligation;
• wherein a reaction solution for the end repair comprises 5-10 μL of 10×NEBuffer 3.1, 1-2 μL of 10 mM 2′-deoxycytidine 5′-triphosphate (dCTP), 1-2 μL of 10 mM 2′-deoxyguanosine 5′-triphosphate (dGTP), 1-2 μL of 10 mM 2′-deoxythymidine 5′-triphosphate (dTTP), 10-20 μL of 1 mM biotin-14- 2′-deoxyadenosine 5′-triphosphate (dATP), and 8-15 μL of 5 U/μL DNA polymerase I large fragment Klenow;
• wherein a reaction solution for the proximity ligation comprises 100-150 μL of 10×T4 ligation buffer, 100-150 μL of 10% TRITON X-100, 40-60 μL of T4 DNA ligase and 300-400 μL of MILLI-Q water.