Cell Culture
S2 cells were cultured in Schneider’s Medium (Gibco, 21720024) supplemented with 10% heat inactivated FBS (Sigma, F7524) and 1% Penicillin/Streptomycin P/S (Sigma, P0781) at 27°C.
K562 cells were incubated in 1× RPMI1640 media supplemented with 10% FBS at 37°C with 5% CO2.
Cell Crosslinking
1. 30 million Drosophila S2 cells or 250 thousand human K562 cells are collected by centrifugation at 300xG for 5 minutes.
2. Resuspend cells in fresh medium. Add freshly made formaldehyde solution (sigma, F8775) to a final concentration of 1%, v/v. Incubate at room temperature for 10 minutes with mixing.
3. Add 1/10 volume of 2.5 M glycine to quench the reaction. Incubate at room temperature for 5 minutes.
4. Centrifuge for 5 minutes at 300xG at 4°C and discard supernatant.
5. Resuspend cells in 1 mL of cold 1X PBS and spin for 5 minutes at 300xG at 4°C.
6. Discard supernatant. The cell pellets can store at -80°C or proceed to the rest of the protocol.
Lysis and Restriction Digest
1. Crosslinked cells were resuspended in 500 μL of ice-cold Hi-C lysis buffer (10 mM Tris-HCl pH8.0, 10 mM NaCl, 0.2% Igepal CA630, 1X protease inhibitors (Sigma, P8340)) and rotated at 4°C for 30 minutes.
2. Nuclei were pelleted at 4 °C for 5 minutes at 2,500xG, and the supernatant was discarded.
3. Pelleted nuclei were washed once with 500 μL of ice-cold Hi-C lysis buffer.
4. Remove supernatant and resuspend pellet with 100 μL of 0.5% SDS. Incubate at 62°C for 10 minutes.
5. Add 285 μL of water and 50 μL of 10% Triton X-100 (Sigma, 93443) to quench the SDS. Mix well, avoiding excessive foaming. Incubate at 37°C for 15 minutes.
6. Add 50 μL of NEB Buffer 3.1 and 20 μL of 10 U/μL DpnII restriction enzyme (NEB, R0147).
7. The sample was rotated at 37°C for 4 h.
8. DpnII was then heat inactivated at 65°C for 20 minutes with no shaking or rotation.
Marking of DNA Ends, Proximity Ligation, and Crosslink Reversal
1. To fill-in the restriction fragment overhangs and mark the DNA ends with biotin, 52 μL of incorporation master mix was then added:
37.5 μL of 0.4 mM biotin–dATP (Thermo Fisher, 19524016);
4.5 μL of dCTP, dGTP, and dTTP mix at 10 mM each (Thermo Fisher, R0151, 10219012, R0161);
and 10 μL of 5 U/μL DNA Polymerase I Large (Klenow) Fragment (NEB, M0210).
2. The reactions were then rotated at 37°C for 45 minutes.
3. Add 948 μL of ligation master mix:
150 μL of 10× NEB T4 DNA ligase buffer with 10 mM ATP (NEB, B0202),
125 μL of 10% Triton X-100,
3 μL of 50 mg/mL BSA (Thermo Fisher, AM2616),
10 μL of 400 U/μL T4 DNA Ligase (NEB, M0202),
and 660 μL of water.
4. The reactions were then rotated at 16 °C for 4 h and room temperature for 1 h.
5. 45 μL of 10% SDS and 55 μL of 20 mg/mL Proteinase K (TRANSGEN, GE201) were added. Incubate at 55°C for at least 2 hours (overnight recommended).
6. Add 20 μL RNase A (TRANSGEN, GE101) and incubate at 37°C for 30 minutes.
7. DNA was purified by phenol:chloroform:isoamyl alcohol (25:24:1) extraction. Dissolve DNA pellet in 100μL of 1X Tris buffer (10 mM Tris-HCl, pH 8)
DNA shearing
1 Shear DNA to a
size of 300-500 bp using the following parameters:
Instrument: Covaris LE220 (Covaris, Woburn, MA)
Volume of Library: 100μL in a Covaris microTUBE
Fill Level: 10
Duty Cycle: 15
PIP: 500
Cycles/Burst: 200
Time: 58 seconds
Biotin Pull-Down and add Full Y-Adaptor
1. Transfer 100 μL of Streptavidin Dynabeads (NEB, S1420S) into a 1.5 mL low-bind tube. Separate on a magnet and discard the solution.
2. Wash the beads with 200 μL 1X Tween Wash Buffer (1X TWB, 5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween-20). Separate on a magnet and discard the solution.
3. Resuspend the beads with 100 μL 2X Binding Buffer ( 2X BB, 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 2M NaCl ).
4. Transfer 100 μL sonicated DNA to the beads resuspended by 2X BB
5. Incubate at room temperature for 15 minutes with rotation to bind biotinylated DNA
to the streptavidin beads.
6. Separate on a magnet and discard the solution.
7. Wash the beads by adding 200 μL of 1X TWB. Heat the tubes on a Thermomixer at 55°C for 2 minutes. Reclaim the beads using a magnet. Discard supernatant.
8. Resuspend beads with 200 μL 1X NEB T4 DNA ligase buffer (NEB, B0202) and transfer to a new 1.5 mL low-bind tube. Separate on magnet and remove the supernatant.
9. Repairing ends of sheared DNA with 100 μL master mix:
10 μL 10X NEB T4 DNA ligase buffer with 10 mM ATP
2 μL of 25 mM dNTP mix
5 μL of 10 U/μL NEB T4 PNK (NEB, M0201)
4 μL of 3 U/μL NEB T4 DNA polymerase I (NEB, M0203)
1 μL of 5 U/μL NEB DNA polymerase I, Large (Klenow) Fragment (NEB, M0210)
78 μL water
10. Incubate at room temperature for 30 minutes.
11. Separate on magnet and remove the supernatant.
12. Add 200 μL 1X TWB to resuspend the beads and incubate on 55°C for 2 minutes. Separate on magnet and remove the supernatant.
13. Add 200 μL 1X NEBuffer 2 to resuspend the beads and transfer to a new 1.5 mL low-bind tube. Separate on magnet and remove the supernatant.
14. Resuspend beads in 100 μL of dATP attachment master mix:
10 μL of 10X NEBuffer 2
5 μL of 10 mM dATP
5 μL of 5U/μL NEB Klenow exo minus (NEB, M0212)
80 μL water
15. Incubate at 37 °C for 30 minutes
16. Separate on magnet and remove the supernatant
17. Add 200 μL 1X TWB to resuspend the beads and incubate on 55°C for 2 minutes. Separate on magnet and remove the supernatant.
18. Add 200 μL 1X Quick ligation reaction buffer (NEB, B6058) to resuspend the beads and transfer to a new 1.5 mL low-bind tube. Separate on magnet and remove the supernatant.
19. Resuspend the beads with 50 μL 1X NEB Quick ligation reaction buffer
20. Add 3 μL Full Y-Adaptor (Vazyme, N802). Mix thoroughly.
21. Add 2 μL of NEB DNA Quick ligase (NEB, M2200).
22. Incubate at room temperature for 30 minutes on rotator.
23. Separate on magnet and remove the supernatant.
Final amplification-free library for sequencing
1. Add 200 μL 1X TWB to resuspend the beads and incubate on 55°C for 2 minutes. Separate on magnet and remove the supernatant.
2. Add 200 μL 0.8X PCR Buffer to resuspend the beads and transfer to a new 1.5 mL low-bind tube. Separate on magnet and remove the supernatant.
3. Resuspend the beads in 100 μL 0.8× PCR Buffer and incubated at 98°C for 10 minutes.
4. Put the tube on wet ice immediately and incubate for 2 minutes.
5. The supernatant was recovered, quantified, and used for direct sequencing on the Illumina HiSeq X10 platform