TITLE: Targeted isolation of circular extrachromosomal DNA by CRISPR-CATCH
AUTHORS: King L Hung, Howard Y. Chang

[DESCRIPTION]
This protocol enables targeted isolation of circular extrachromosomal DNA using CRISPR-Cas9-mediated linearization followed by pulsed field gel electrophoresis. Isolated DNA products can be subsequently used for short-read sequencing, single-molecule long-read sequencing or other assays.

[GUIDELINES]
Note 1 - PFGE parameters:

Settings for DNA 0.22-2.2Mb, 0.5X TAE, 14C, 1% LMP agarose, linear ramping. 
Two-state mode
Run time duration: 16:39
Initial switch time: 0:00:20.16
Final switch time: 0:02:55.12
Gradient: 6V/cm
Included angle: 120
Ramping: 0.0 (linear)

Settings for DNA 1-6Mb, 0.5X TAE, 14C, 0.8% LMP agarose, linear ramping. 
Multi-state mode
Block 1:
Duration: 59:02
initial switch time: 0:20:00
final switch time: 0:40:24
voltage gradient: 2V/cm
ramping: 0.0 (linear)
angle: -53 (state 1), +53 (state 2)
Block 2:
Duration: 9:15
initial switch time: 0:01:10.2
final switch time: 0:02:09.92
voltage gradient: 6V/cm
ramping: 0.0 (linear)
angle: -60 (state 1), +60 (state 2)

Electro-depletion for removal of short linear DNA from agarose plugs.
Block 1:
duration: 3:00:00
initial switch time: 3:00:00
final switch time: 3:00:00
voltage gradient: 5.2V/cm
ramping: 0.0 (linear)
angle: 0 (state 1)
Block 2:
duration: 0:02:00
initial switch time: 0:02:00
final switch time: 0:02:00
voltage gradient: 5.2V/cm
ramping: 0.0 (linear)
angle: 180 (state 1)

[STEPS]
SECTION: Embedding cells in agarose plugs
1. Melt 1% Certified Low Melt Agarose solution in PBS, place in 45C water bath.
SECTION: Embedding cells in agarose plugs
2. Pellet 1 million cells per agarose plug at 300g for 5 minutes. (for example, spin down 10 million cells to make 10 agarose plugs)
SECTION: Embedding cells in agarose plugs
3. Two cold PBS washes (30ml) with gentle pipet mixing. Pellet cells after each wash at 300g for 5 minutes.
SECTION: Embedding cells in agarose plugs
4. Resuspend cells at 1 million cells per 30 ul PBS using a wide-bore pipet tip. Warm to 37C.
SECTION: Embedding cells in agarose plugs
6. Mix with equal volume 1% Low Melt Agarose (pre-cooled to 37-45C) using a wide-bore pipet tip. Immediately pipet into plug mold without introducing bubbles.
SECTION: Embedding cells in agarose plugs
7. Cool samples at 4C for 10 minutes.
SECTION: Embedding cells in agarose plugs
8. Transfer each agarose plug into an eppendorf tube. Add 1ml SDE and incubate at room temperature for 10min with gentle shaking.
SECTION: Embedding cells in agarose plugs
9. Remove supernatant without breaking the agarose plug. Add 1ml ES and incubate at 50C for 1-2 days with gentle shaking (~400rpm).
SECTION: Embedding cells in agarose plugs
10. Cool briefly on ice.
SECTION: Embedding cells in agarose plugs
11. Remove supernatant, add 1ml of 25mM EDTA containing 1mM PMSF (dilute PMSF right before use) to inactivate proteinase K. Incubate at RT for 1 hour on a shaker.
SECTION: Embedding cells in agarose plugs
12. Remove supernatant, add 1ml of 25mM EDTA with 1mg/ml RNase A, incubate at 37C for 30min.
SECTION: Embedding cells in agarose plugs
13. Remove supernatant, wash with 1ml of 25mM EDTA with a 5min incubation.
SECTION: Embedding cells in agarose plugs
14. Remove supernatant, add 1ml of 25mM EDTA and store at 4C (2-12 months).
SECTION: ecDNA linearization by CRISPR-Cas9
25. Set up a reaction mix in the following order:

 
Incubate reaction mix at room temperature for 10 minutes.
SECTION: ecDNA linearization by CRISPR-Cas9
24. Remove supernatant from agarose plug, 3 washes with 300ul 1X NEBuffer 3.1 with 5min incubations. 
SECTION: ecDNA linearization by CRISPR-Cas9
26. Remove supernatant from agarose plug. Add reaction mix to agarose plug. 
SECTION: ecDNA linearization by CRISPR-Cas9
27. Incubate at 37C for 4 hours on a shaker with gentle shaking (~400-500 rpm). 
SECTION: ecDNA linearization by CRISPR-Cas9
28. Remove supernatant. Add 200ul 25mM EDTA with 3ul proteinase K, mix, incubate at RT overnight with gentle shaking (~400-500 rpm). 
SECTION: ecDNA linearization by CRISPR-Cas9
29. Remove supernatant. Add 1ml 25mM EDTA with 1mM PMSF, incubate at RT for 1 hour with gentle shaking.
SECTION: ecDNA linearization by CRISPR-Cas9
30. (for storage) Remove supernatant, do two washes in 25mM EDTA, resuspend in 25mM EDTA and store at 4C. (Skip to next step if running PFGE immediately) 
SECTION: ecDNA separation by pulsed field gel electrophoresis (PFGE)
31. Make a PFGE gel using 1% Certified Low Melt Agarose in 0.5X TAE buffer. Do not add stain yet. Gently remove the gel comb to avoid destroying the integrity of the wells.
Note: agarose type, percentage, and buffer type all depend on the objective. See Note 1 under Guidelines for recommendations.
SECTION: ecDNA separation by pulsed field gel electrophoresis (PFGE)
35. Refill electrophoresis chamber with 2.2 liters of running buffer. Turn pump knob to 70, and turn on cooling module, in this order. Set temperature to proper temperature (typically 14C; see Note 1 in Guidelines) on cooling module. 
SECTION: ecDNA separation by pulsed field gel electrophoresis (PFGE)
36. Equilibrate DNA ladder agarose plugs and samples in running buffer (2X washes with 5min incubations). 
SECTION: ecDNA separation by pulsed field gel electrophoresis (PFGE)
37. Load DNA ladders into wells using a microscope cover slip. Push plug all the way to the bottom of the well to make sure there is no space. 
SECTION: ecDNA separation by pulsed field gel electrophoresis (PFGE)
38. Load DNA samples in agarose plugs into wells. Seal all wells (including empty ones) with molten 1X Low Melt Agarose. Wait ~10min for agarose to solidify. Carefully place gel in the electrophoresis chamber. 
SECTION: ecDNA separation by pulsed field gel electrophoresis (PFGE)
39. Start electrophoresis using appropriate settings based on size range, agarose type, percentage, and buffer type. (See Note 1 in Guidelines)
SECTION: ecDNA separation by pulsed field gel electrophoresis (PFGE)
41. Transfer the gel carefully to a container. Stain gel in 3X gelred with 0.1 M NaCl on rocker for 30min. Cover with aluminum foil to protect from light.
SECTION: ecDNA separation by pulsed field gel electrophoresis (PFGE)
42. Rinse gel with water.
SECTION: ecDNA separation by pulsed field gel electrophoresis (PFGE)
43. Take an image of the gel.
SECTION: ecDNA separation by pulsed field gel electrophoresis (PFGE)
44. Excise bands using a clean cover glass based on relative distances to notches on the side.
SECTION: Extraction of high molecular weight DNA molecules after PFGE
45. Wash agarose twice with 2 volumes or 500ul  of 1X agarase buffer on ice for 10-30min each.
SECTION: Extraction of high molecular weight DNA molecules after PFGE
46. Remove buffer, melt agarose at 65C for 10min.
SECTION: Extraction of high molecular weight DNA molecules after PFGE
47. Incubate agarose at 43C for 10min.
SECTION: Extraction of high molecular weight DNA molecules after PFGE
48. Add 1U agarase per 200ul agarose, incubate at 43C for 1 hour with shaking (400rpm).
SECTION: Extraction of high molecular weight DNA molecules after PFGE
49. Add 0.1 volume 3M sodium acetate. Chill on ice for 15 minutes.
SECTION: Extraction of high molecular weight DNA molecules after PFGE
50. Centrifuge at 15,000g for 15 minutes at 4C to pellet any remaining undigested carbohydrates.
SECTION: Extraction of high molecular weight DNA molecules after PFGE
51. Remove the DNA-containing supernatant. Add 2 volumes of isopropanol. 
SECTION: Extraction of high molecular weight DNA molecules after PFGE
52. Mix, chill at 4C for 10min, centrifuge at 15,000g for 15 minutes at 4C.
SECTION: Extraction of high molecular weight DNA molecules after PFGE
53. Remove supernatant, wash the pellet with 500ul ice-cold 70% ethanol. Pipet up and down to solubilize salt in the pellet. Pellet DNA at max speed for 5min at 4C.
SECTION: Extraction of high molecular weight DNA molecules after PFGE
54. Remove supernatant. Do a second 70% ethanol wash if pellet looks like it has excessive salt. 
SECTION: Extraction of high molecular weight DNA molecules after PFGE
55. Re-pellet, use a small pipet tip to remove residual ethanol.
SECTION: Extraction of high molecular weight DNA molecules after PFGE
56. Air-dry for 5-10min. Do not over-dry. Resuspend in EB buffer.
SECTION: ecDNA separation by pulsed field gel electrophoresis (PFGE)
32. Clean electrophoresis chamber with 2 liters of 10% bleach. Turn on power module, power outlet to pump, turn pump knob to 100 to allow bleach to circulate for 30 minutes. 
SECTION: ecDNA separation by pulsed field gel electrophoresis (PFGE)
33. Remove and completely drain bleach from the system. Add 2 liters of DI water and turn on pump to allow water to circulate. Drain water and repeat for a total of 3 washes.
SECTION: ecDNA separation by pulsed field gel electrophoresis (PFGE)
34. Remove and drain water from the system. Add 2 liters of running buffer (composition depends on PFGE parameters; typically 0.5X TAE. See Note 1 in Guidelines) and turn on pump to allow buffer to circulate. Drain buffer.  
SECTION: Embedding cells in agarose plugs
5. Place plug molds on ice.
SECTION: ecDNA separation by pulsed field gel electrophoresis (PFGE)
40. Make notches on the side of the gel using glass cover slips to mark distances. 
SECTION: Removal of degraded DNA fragments (optional)
15. For samples with high dead-cell fractions, samples that were previously frozen, or tumor samples with degraded DNA due to the sample preparation process, this optional section allows background depletion for a cleaner gel and improved ecDNA enrichment. This is usually not necessary for cell lines and high-quality cell samples, but may be necessary for primary tumor samples. 
SECTION: Removal of degraded DNA fragments (optional)
16. Make a PFGE gel using 1% Certified Low Melt Agarose in 0.5X TAE buffer. Do not add stain. Gently remove the gel comb to avoid destroying the integrity of the wells.
SECTION: Removal of degraded DNA fragments (optional)
17. Clean electrophoresis chamber with 2 liters of 10% bleach. Turn on power module, power outlet to pump, turn pump knob to 100 to allow bleach to circulate for 30 minutes. 
SECTION: Removal of degraded DNA fragments (optional)
18. Remove and completely drain bleach from the system. Add 2 liters of DI water and turn on pump to allow water to circulate. Drain water and repeat for a total of 3 washes.
SECTION: Removal of degraded DNA fragments (optional)
19. Remove and drain water from the system. Add 2 liters of running buffer and turn on pump to allow buffer to circulate. Drain buffer. 
SECTION: Removal of degraded DNA fragments (optional)
20. Refill electrophoresis chamber with 2.2 liters of running buffer. Turn pump knob to 70, and turn on cooling module, in this order. Set temperature to proper temperature (typically 14C; see Note 1 in Guidelines) on cooling module. 
SECTION: Removal of degraded DNA fragments (optional)
21. Load DNA samples in agarose plugs into wells. Do NOT seal wells with agarose. Carefully place gel in the electrophoresis chamber. 
SECTION: Removal of degraded DNA fragments (optional)
22. Start electrophoresis using the electro-depletion settings. (See Note 1 in Guidelines, "Electro-depletion for removal of short linear DNA from agarose plugs"; if very large >1Mb DNA fragments are observed, the settings for DNA 0.22-2.2Mb may also be used for electro-depletion)
SECTION: Removal of degraded DNA fragments (optional)
23. Transfer the gel carefully to a container. Make two incisions in each upper corner of the well to remove the part of the gel above the well, exposing the plug sitting inside. Carefully take out the plug without damaging it. Place the plug in an eppendorf tube, add 1ml of 25mM EDTA and store at 4C (2-12 months). This plug contains intact genomic DNA from the original sample but is depleted of degrade DNA fragments. This plug can be used subsequently for CRISPR-CATCH.