TITLE: DISCOVER-Seq: MRE11 ChIP Seq
AUTHORS: Lilly van de Venn, Zacharias Kontarakis, Beeke Wienert

[DESCRIPTION]
Genome editing using nucleases such as CRISPR-Cas induces programmable DNA damage at a target genomic site, but can also affect off-target sites. We develop a powerful assay for the unbiased identification of off-target sites that we term DISCOVER-seq. This approach takes advantage of the recruitment of endogenous DNA repair factors for genome-wide identification of Cas-induced double strand breaks. One such factor, MRE11, is recruited so precisely to a double stranded break that nuclease cut sites can be determined with single-base resolution. DISCOVER-seq is applicable to multiple types of Cas nucleases and provides an unprecedented molecular picture of events that precede repair of the affected sites. DISCOVER-seq furthermore detects off-targets in cellular models and tissues.

[STEPS]
SECTION: RNP editing of cells
1. Culture your cell line of interest so that you can edit 1x10^7 cells on the day of nucleofection. 
 

 

SECTION: RNP editing of cells
2. Harvest cells and spin down in falcon tube. 
SECTION: RNP editing of cells
3. Wash cells with 1ml PBS and transfer to eppendorf tube. Spin to pellet, discard S/N
SECTION: RNP editing of cells
4. While cells are spinning: Prepare RNPs. 

Add Cas9 Buffer (20 mM HEPES (pH 7.5), 150 mM KCl, 1 mM MgCl2, 10% glycerol and 1 mM TCEP) such that final volume of all components comes to 15uL.
Add 30 pmol of Cas9 per one million cells (i.e. for 1x10^7 you need 300pmol Cas9)
Add 30 pmol of sgRNA per one million cells (i.e. for 1x10^7 you need 300pmol gRNA)
Incubate at RT for 5-10 mins (or longer)
SECTION: RNP editing of cells
5. Add 60ul of nucleofection buffer (Lonza) including supplements to your RNPs and pipette up and down. Transfer the whole volume into eppendorf tube with cell pellets and resuspend cells. 
 

 

SECTION: RNP editing of cells
6. Transfect cell suspension into 100ul nucleofection cuvette and electroporate using the a Lonza 4D X nucleofector. 
 

SECTION: RNP editing of cells
7. After nuclefection, let cells sit at RT in cuvette for 5 mins. Then transfer cells into culture plates with prewarmed complete media and incubate at 37C and 5% CO2. 
SECTION: Crosslinking
8. After 8-24h of culture, harvest cells into falcon tube. Spin to pellet. Discard media.

IMPORTANT: Before you spin cells down, make sure to take a small split of the cells to keep in culture for a few more days. You will need these cells to later extract gDNA to check on mutation rates at off-target sites. We usually harvest cells for gDNA extraction 4 days post nucleofection.
SECTION: Crosslinking
9. Resuspend cells in 20ml of room temperature media (You can use IMDM or RPMI) without supplements. 
Add 37% Formaldehyde to a final concentration of 1%. Incubate at RT for 15 min

 


 

SECTION: Crosslinking
10. Add 2.5 M glycine to quench formaldehyde (final 125mM). Leave a couple of minutes at RT. 
 

SECTION: Crosslinking
11. Wash cells twice with 10 ml ice cold PBS (1200 g, 3 min spins – pour off S/N each time). Transfer cells to a 15 mL falcon tube before last spin. Take off S/N completely.
SECTION: Crosslinking
12. Proceed with lysis, or snap freeze cells in liquid nitrogen and store pellets at −80 °C.
Crosslinked cell pellets can be stored at -80C for a few months.
SECTION: Prebinding of antibody to magnetic beads
13. 
 

Add 100 μl magnetic beads (Invitrogen, Dynabeads) – protein A to 1.5 ml microfuge tubes. Add 1 ml block solution (0.5% BSA (w/v) in PBS). If you have multiple IPs you can scale up accordingly to the number of IPs. 

e.g.: 3 IPs: use 300ul of beads (still wash with 1ml of Block solution)

 

SECTION: Prebinding of antibody to magnetic beads
14. Collect the beads using magnetic stand. Remove supernatant. Wash beads in 1ml block solution two more times. 

 

SECTION: Prebinding of antibody to magnetic beads
15. Resuspend beads in 250ul block solution per IP and add 4ul of MRE11 antibody (NB100-142). 

e.g.: 3 IPs: use 750ul of block solution
 

SECTION: Prebinding of antibody to magnetic beads
16. Incubate for 1-2 hours at on a rotating platform @ RT. 
SECTION: Prebinding of antibody to magnetic beads
17. When ready to IP: Wash magnetic beads as described above (3 times in 1 ml block solution). Resuspend in 100 μl block solution which is to be added to lysate (Step 25). 

SECTION: Cell lysis
18. Resuspend each pellet of crosslinked cells in 10 ml of LB1 (+protease inhibitors) (50 mM Hepes–KOH, pH 7.5; 140 mM NaCl; 1 mM EDTA; 10% Glycerol; 0.5% NP-40 or Igepal CA-630; 0.25% Triton X-100). Rock at 4 °C for 10 min. 

 

SECTION: Cell lysis
19. Spin at 2000 × rcf for 3 min at 4 °C in a tabletop centrifuge. Pour off S/N.
SECTION: Cell lysis
20. Resuspend each pellet in 10 ml of LB2 (+protease inhibitors)  (10 mM Tris–HCL, pH8.0; 200 mM NaCl; 1 mM EDTA; 0.5 mM EGTA). Rock gently at 4 °C for 5 min. 
SECTION: Cell lysis
21. Pellet nuclei in tabletop centrifuge by spinning at 2000 × rcf for 3 min at 4 °C. Pour off S/N
SECTION: Cell lysis
22. Resuspend each pellet in each tube in 1.5 ml LB3 (+protease inhibitors) in 2mL tube (10 mM Tris–HCl, pH 8; 100 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 0.1% Na–Deoxycholate; 0.5% N-lauroylsarcosine).
SECTION: Cell lysis
23. Sonicate lysate 3x for 1 min on ice. Settings: 0.9 ON 0.1 OFF and 75% amplitude works for our sonicator.  Let samples rest on ice inbetween sonications. 

 

 

SECTION: Cell lysis
24. Spin at 20,000 × rcf for 10 min at 4 °C to pellet debris. Transfer S/N to new falcon tube. Add 1.5ml of LB3 and 300ul of 10% Triton-X (final 1%). 


 

SECTION: Cell lysis
25. IMPORTANT: Save 2x 50 μl of cell lysate from each sonication as input DNA in screw cap tubes. Store at −20 °C until the next day.

Now you are ready for IP. 
SECTION: Chromatin IP
26. Add 100 μl antibody/magnetic bead mix from  to cell lysates. 

SECTION: Chromatin IP
27. Gently mix on rotator or rocker at 4 °C overnight.
SECTION: Wash beads and elution
28. Transfer half the volume of an IP to an eppendorf tube.
SECTION: Wash beads and elution
29. Let tubes sit in magnetic stand to collect the beads. Remove supernatant and add remaining IP. Let tubes sit again in magnetic stand to collect the beads and remove supernatant.
SECTION: Wash beads and elution
30. Add 1 ml RIPA Buffer (50 mM Hepes–KOH, pH 7.5; 500 mM LiCl; 1 mM EDTA; 1% NP-40 or Igepal CA-630; 0.7% Na–Deoxycholate) to each tube. Turn tubes to wash beads. Collect beads. Remove supernatant. 
SECTION: Wash beads and elution
31. Repeat this wash 5 more times. 
SECTION: Wash beads and elution
32. Wash once with 1 ml TBS (20 mM Tris–HCl, pH 7.6; 150 mM NaCl). Remove S/N.
SECTION: Wash beads and elution
33. Add 200 μl of elution buffer (50 mM Tris–HCl, pH 8; 10 mM EDTA; 1% SDS). Transfer to 1.5ml conical screw cap tubes.
SECTION: Wash beads and elution
34. Elute and perform reverse crosslinking at 65 °C for 6–18 h rotating or shaking. 
SECTION: Wash beads and elution
35. Thaw 50 μl of the input lysate from  , add 150 μl of elution buffer and mix. Reverse the formaldehyde crosslinking as in   simultaneously with the ChIP samples.


SECTION: DNA purification
36. Quickspin tubes to collect liquid from lids. Collect beads with magnetic stand. Take 200 μl of supernatant and transfer to a new tube.
SECTION: DNA purification
37. Add 100 μl of TE to each tube of IP and input DNA to dilute SDS in elution buffer.
SECTION: DNA purification
38. Add 8 μl of 1 mg/ml RNaseA (Ambion Cat # 2271). Mix and incubate at 37 °C for 30 min.

 

SECTION: DNA purification
39. Add 4 μl of 20 mg/ml proteinase K (Invitrogen, 25530-049). Mix and incubate at 55 °C for 1h.

 

SECTION: DNA purification
40. Purify DNA using a Qiagen clean up kit. 

Make sure to check on pH of your reaction by using the provided pH indicator. Add 5ul 3M Sodium Acetate, pH 5.2 to adjust pH if necessary.
 

 
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SECTION: DNA purification
41. Elute DNA in 41ul of purfied water. 



SECTION: QC of pulldown
42. Check if ChIP was successful by amplifying a region close to your on-target site (about 200bp from cut site) by quantitative real-time PCR. 
Also run your input DNA to account for differences in primer efficiencies and amount of starting material.

Compare the Cts obtained in this region to a random region elsewhere in the genome. 

For qPCR we dilute input DNA 1 in 49 and IP DNA 1 in 5.

 


SECTION: Illumina Sequencing
43. If the qPCR was successful proceed to library preparation for Illumina sequencing. 

We sequence our samples using 100bp paired-end sequencing on a HiSeq4000 aiming for 12.5 Million reads per sample.
SECTION: BLENDER bioinformatics pipeline
44. Analyze your sequencing data using BLENDER software which is available on GitHub. https://github.com/cornlab/blender
SECTION: Amplicon-NGS for off-targets
45. Don't forget to harvest your cells from  for genomic DNA extraction. 
Check sites identified by BLENDER for indels using amplicon next-generation sequencing.