Chromatin immunoprecipitation (ChIP) protocol for the sponge Amphimedon queenslandica

1. Squeeze approx. 1cm³ of adult material through a fine cloth/syringe with mesh. 
2. For larval material, pool ~350 together and homogenize
3. Collect the liquid into a falcon tube
4. Bring volume to 10mL with Filtered Sea Water (FSW) (0.22um) 
5. Add formaldehyde to a final concentration of 2%, mix and incubate for 5-10 minutes at RT 
6. Add 2M glycine to final concentration of 0.125M to quench the cross-linking, mix and incubate for 5 minutes on RT/ice
7. Wash 2x for 5 min each in cold 0.22 µm filtered seawater (around 500g spin)
8. Resuspend the cell pellet in 500uL of SDS Lysis Buffer to which 1X protease and phosphatase inhibitor has been added and incubate for at least 10 minutes on ice. Avoid foaming.
9. Sonicate lysate to shear DNA to lengths between 200 and 500 bp on Bioruptor Diagenode (12 cycles of 30s ON, 30s OFF, high voltage). Avoid over-heating adding ice to the machine after 6 cycles. Sonication conditions should be optimized for each experiment, especially when using a different Bioruptor.
10. Centrifuge lysate for 10 minutes at 12,000g at 4°C. Remove supernatant and discard pellet. The supernatant has to be stored at -80C, avoid many freeze/thaw cycles and keep on ice once thawed. Remove 50uL (1/10 of initial volume) as ‘INPUT’ DNA and store it at -20°C. 
11. Use 50uL of lysate per IP. Dilute the sonicated extract 10 fold in ChIP Dilution Buffer + PhosSTOP phosphatase inhibitor and cOmplete protease inhibitor cocktail. For example, add 450uL ChIP Dilution Buffer to 50uL of lysate per IP. For 3 IPs, add 1350uL of ChIP Dilution Buffer to 150uL of lysate.
12. Preclear chromatin with beads
    1. Use 10uL of Dynabeads (usually protein G) per IP and wash twice with 10uL of ChIP Dilution buffer
    2. Use magnets for 2 min each capture, then aspirate supernatant
    3. Add the 500uL of chromatin from step 10 to the 10uL of beads.
    4. Rotate at 4°C for 1 hour.
13. Link beads with antibody 
    1. Wash 20uL of Dynabeads (usually Protein G) per IP and wash twice with 20uL of the appropriate binding buffer. 
       1. Binding buffer for protein G is 0.1M Na-Citrate pH 5
       2. Binding buffer for Protein A is 0.1M Na-phosphate buffer pH 8
       3. Check the affinity of Protein A and/or G to your specific IgG; e.g. Protein A/G are not suitable for chicken IgY. 
    2. Use magnets for 2 min each capture, then aspirate supernatant.
    3. Resuspend beads in 20uL of the appropriate binding buffer
    4. Add 5uL of each antibody per IP. Add no antibody to Moch control.
       1. The amount of antibody should be optimized for each experiment and each antibody.
    5. Rotate at 4°C for 1 hour. 
14. Collect the supernatant fraction from step 11 and discard beads
15. Capture beads from step 12 and discard supernatant
16. Add chromatin fraction from step 13 to beads from step 14
17. Rotate overnight at 4°C.
18. Use magnets for 2 min to capture beads. Remove supernatant. 
19. Wash the beads using the procedure and sequence of buffers below. Perform each of the 3 washes with 800uL of cold buffers with gentle agitation. To be on the safe side, PhosSTOP phosphatase inhibitor and cOmplete protease inhibitor cocktail can be added to the wash buffers. Alternatively, washes can be performed at 4C. After each wash use magnets to capture the beads. 
    1. Low Salt Wash Buffer (30s, 5 min, 5 min)
    2. High Salt Wash Buffer (30s, 5 min, 5 min)
    3. LiCl Wash Buffer (30s, 5 min, 5 min)
    4. TE buffer (30s, 5 min, 5 min). 
20. Remove as much TE as possible after last wash
21. To elute the immune complex from the washed beads, add 175uL TES (alternatively use Elution Buffer) to each sample. Mix by pipetting and incubate at RT for 5 min with gentle rotation. Incubate at 65°C for additional 15 min with constant flicking to avoid beads precipitation. 
22. Use magnets to recover the beads and transfer supernatant to a fresh tube (keep this on ice for the time being).
23. Repeat Steps 20-21. Combine the eluates. You should now have 350uL of eluate per IP. 
24. Add 14uL of 5M NaCl and reverse the cross-linking by incubating at 65°C overnight with gentle shaking (~100 rpm). Include also the ‘INPUT’ DNA from step 9; add TES to final volume of 350uL and add 14uL of 5M NaCl and incubate overnight at 65°C. 
25. To each sample add 3.5uL of RnaseA and incubate at 37°C for 1 hour.
26. Add 7uL of 0.5M EDTA, 14uL of 1M Tris-HCl pH6.5, 2uL of 20mg/mL Proteinase K and incubate for 1 hour at 45°C.
27. Extract DNA with 1x phenol/chloroform, 1x chloroform extraction, overnight precipitation at -20°C (or at least a few hours at -80°C) with 2.5 volumes ice-cold 100% ethanol, 2uL glycoblue and 1/10 volume of salt. Centrifuge at max speed at 4°C for 15 minutes. Discard supernatant. Wash the DNA pellets with ice-cold 70% ethanol. Centrifuge with same condition as above. Air-dry the pellet for 5 minutes (DO NOT OVERDRY). Resuspend the pellet in 15uL of sterile RNAse and DNase free water. 
    1. Alternatively, QIAGEN PCR purification kit can be used to extract DNA. 
28. Store the samples at -20°C until ready for step 28. 
29. Proceed with library preparation for NGS. 

Buffers and Reagents

SDS Lysis Buffer

1% SDS

10 mM EDTA

50mM Tris-HCl pH 8

add protease and phosphatase inhibitors before use

ChIP Dilution Buffer

0.01% SDS

1.1% Triton X-100

1.2 mM EDTA

16.7 mM Tris-HCl pH 8

167 mM NaCl

add PI before use

Low Salt Wash Buffer

0.1% SDS

1% Triton X-100

2 mM EDTA

20 mM Tris-HCl pH 8

150mM NaCl

High Salt Wash Buffer

0.1% SDS

1% Triton X-100

2mM EDTA

20mM Tris-HCl pH 8

500mM NaCl

LiCl Wash Buffer

0.25M LiCl

1% NP-40

1% deoxycholic acid (sodium salt) aka D.O.C.

1mM EDTA

1mM Tris-HCl pH 8

TE

10mM Tris pH 8 and 1mM EDTA

TES 

TE + 0.1% SDS

Elution Buffer

1%SDS

0.1M NaHCO₃

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