DISCOVER-Seq: MRE11 ChIP Seq

Culture your cell line of interest so that you can edit 1x10^7 cells on the day of nucleofection.

Harvest cells and spin down in falcon tube.

Wash cells with 1ml PBS and transfer to eppendorf tube. Spin to pellet, discard S/N

While cells are spinning: Prepare RNPs.

1. 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 .
2. Add 30 pmol of Cas9 per one million cells (i.e. for 1x10^7 you need 300pmol Cas9)
3. Add 30 pmol of sgRNA per one million cells (i.e. for 1x10^7 you need 300pmol gRNA)
4. Incubate at RT for 5-10 mins (or longer)

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.

Transfect cell suspension into 100ul nucleofection cuvette and electroporate using the a Lonza 4D X nucleofector.

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.

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.

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

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

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.

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.

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)

Collect the beads using magnetic stand. Remove supernatant. Wash beads in 1ml block solution two more times.

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

Incubate for 1-2 hours at on a rotating platform @ RT.

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).

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 .

Spin at 2000 × rcf for 3 min at 4 °C in a tabletop centrifuge. Pour off S/N.

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 .

Pellet nuclei in tabletop centrifuge by spinning at 2000 × rcf for 3 min at 4 °C. Pour off S/N

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).

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.

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%).

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.

Add 100 μl antibody/magnetic bead mix from to cell lysates.

Gently mix on rotator or rocker at 4 °C overnight.

Transfer half the volume of an IP to an eppendorf tube.

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.

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.

Repeat this wash 5 more times.

Wash once with 1 ml TBS (20 mM Tris–HCl, pH 7.6; 150 mM NaCl). Remove S/N.

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.

Elute and perform reverse crosslinking at 65 °C for 6–18 h rotating or shaking.

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.

Quickspin tubes to collect liquid from lids. Collect beads with magnetic stand. Take 200 μl of supernatant and transfer to a new tube.

Add 100 μl of TE to each tube of IP and input DNA to dilute SDS in elution buffer.

Add 8 μl of 1 mg/ml RNaseA (Ambion Cat # 2271). Mix and incubate at 37 °C for 30 min.

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

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.

Elute DNA in 41ul of purfied water.

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.

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.

Analyze your sequencing data using BLENDER software which is available on GitHub. https://github.com/cornlab/blender

Don't forget to harvest your cells from for genomic DNA extraction.

Check sites identified by BLENDER for indels using amplicon next-generation sequencing.