Immunoprecipitation of Target Protein-DNA Complexes from Motor Neuron Lysate

Protokoll

NOTE: Autoclaved distilled and deionized water (ddH₂O) is recommended for making buffers and reaction master mixes.

1. Harvesting and crosslinking cells

1. After differentiating mouse embryonic stem (ES) cells into postmitotic neurons, add 11% formaldehyde to the harvested cells to a final concentration of 1% (v/v). Rock cells on a rocking platform (a rocker, shaker, or rotator) for 15 min, at room temperature (RT, 25 °C).
   NOTE: Depending on the target protein to be crosslinked, less formaldehyde crosslinking (for example, a final concentration of 0.5%) or double crosslinking with disuccinimidyl glutarate (DSG) can be used.
2. Add 2.5 M glycine to a final concentration of 150 mM to stop the crosslinking reaction. Rock cells on a rocking platform at RT, for 5 min.
3. Centrifuge the crosslinked cells in 15 mL conical tubes at RT, for 6 min, at 1,350 x g. Aspirate the solution, then resuspend cells in 5 mL of 1x phosphate-buffered saline (PBS).
   NOTE: The crosslinked cell pellets can be stored at -80 °C after flash freezing with liquid nitrogen.

2. Cell lysis

NOTE: The following steps in this protocol are for approximately 2 x 10⁷ neuronal cells differentiated from mouse ES cells. To break open cells, lysis buffers containing various detergents will be used. Add 50 µL of 1000x complete protease inhibitor (CPI) stock to 50 mL of buffer just prior to use.

1. Prepare lysis buffers 1−3 as described in Table 1.
2. Thaw crosslinked cell pellets on ice, then thoroughly resuspend cell pellets in 3 mL of lysis buffer 1 in 15 mL conical tubes. Rock at 4 °C for 15 min on a rocking platform. Centrifuge at 1,350 x g for 5 min at 4 °C and aspirate supernatant.
3. Thoroughly resuspend pelleted cells in 3 mL of lysis buffer 2. Rock at 4 °C for 10 min on a rocking platform. Centrifuge at 1,350 x g for 5 min at 4 °C and aspirate supernatant.
4. Add 1 mL of lysis buffer 3 to each pellet, then keep on ice. Immediately proceed to sonication.

3. Sonicating chromatin

NOTE: Keep samples on ice or at 4 °C during this sonication procedure to reduce crosslink reversal.

1. Using a sterile spatula, add sonication beads (Table of Materials) up to the 0.2 mL graduation mark on 15 mL polystyrene tubes. Wash sonication beads by vortexing in 600 µL of 1x PBS until no dry spots are visible. Centrifuge the tubes for 5 s at 30 x g and aspirate 1x PBS.
   NOTE: Sonication in polystyrene tubes is more efficient than sonication in polypropylene tubes. If a smaller number of cells (for example, <10⁶ cells) is sonicated, use 1.5 mL polystyrene tubes without adding sonication beads.
2. Thoroughly resuspend the nuclear lysates in 1 mL of lysis buffer 3 (from step 2.4), then transfer to the 15 mL polystyrene tubes containing sonication beads. Briefly vortex the polystyrene tubes.
3. To fragmentize the crosslinked chromatin DNA, sonicate nuclear lysates at 4 °C for an optimized number of cycles, with power amplitude at 30 W, and sonication cycles set to 30 s on/30 s off.
   NOTE: Optimization of sonication for each cell type and batch will result in the best chromatin immunoprecipitation-exonuclease (ChIP-exo) yield. For 2 x 10⁷ mouse neuronal cells, 20−30 sonication cycles at the mentioned settings will fragment the chromatin in the range of 100−500 bp.
4. After sonication is complete, transfer all the supernatant (sonicated lysates), from each sample, to 1.5 mL tubes. Add 10% 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol) to each sample at a final concentration of 1%. Mix by pipetting.
5. To pellet cell debris, centrifuge samples at 13,500 x g for 10 min at 4 °C. Transfer all the sonicated lysates (supernatant) from each sample to new 1.5 mL tubes.
6. Take 30 µL of sonicated lysate from each sample (~3% of sonicated lysate) and add to new 1.5 mL tubes to check sonication. Store remaining sonicated lysates at 4 °C until ready for incubation with antibody-coated beads.

4. Checking sonication

1. Make 20 mL of 2x proteinase K buffer by adding 2 mL of 0.5 M Tris-HCl (pH 8.0), 2 mL of 0.5 M ethylenediamine tetraacetic acid (EDTA), 10 mL of 10% sodium dodecyl sulfate (SDS) and 6 mL of autoclaved ddH₂O. Do not add CPI to this buffer. Store in 50 mL tube at RT.
2. To reverse the protein-DNA crosslink, by removing the proteins, take the 30 µL of sonicated chromatin from each sample (from step 3.6), add 166 µL of autoclaved ddH₂O, 200 µL of 2x proteinase K buffer and 4 µL of 20 mg/mL proteinase K. Briefly vortex the samples, and then incubate at 65 °C for 1−3 h at 24 x g.
3. NOTE: The sonicated lysates can be reverse crosslinked at 65 °C overnight.
4. Extract DNA using phenol:chloroform:isoamyl alcohol (PCIA: 25:24:1) and ethanol precipitation method as follows.
   CAUTION: PCIA is toxic. Use under a fume hood with standard personal protective equipment (PPE).
   1. Add 400 µL of PCIA to each sample in 1.5 mL tubes, set vortex to maximum speed and vortex samples for 20 s. Centrifuge at 18,400 x g for 6 min at RT. Two phases will be observed. Carefully transfer the upper aqueous layer (clear phase) of each sample to new 1.5 mL tubes.
   2. Add 1 µL of 10 mg/mL RNase A. Incubate samples at 37 °C for 30 min.
   3. Add 1 µL of 20 mg/mL glycogen to each sample, then precipitate with 1 mL of ice-cold 100% ethanol (stored in a -20 °C freezer). Mix briefly, then incubate samples in -80 °C freezer for 30 min to 1 h. Centrifuge samples at 18,400 x g for 10 min at 4 °C and carefully pour out 100% ethanol.
   4. Wash pellets with 500 µL of ice-cold 70% ethanol (stored in a -20 °C freezer). Centrifuge at 18,400 x g for 5 min at 4 °C. Carefully pour out 70% ethanol.
   5. Incubate samples in 1.5 mL tubes at 50 °C until the remaining ethanol is evaporated. Resuspend DNA pellets in 15 µL of autoclaved ddH2O or nuclease-free ddH2O.
   6. Run the extracted DNA samples, along with a DNA ladder, in a 1.5% agarose gel at 120−180 V and check the size of the sonicated DNA.

5. Antibody incubation with beads

NOTE: The following steps in this protocol are for approximately 2 x 10⁷ neuronal cells differentiated from mouse ES cells. Do not freeze and thaw magnetic beads at any point during the ChIP-exo protocol as the beads may crack causing contamination of the sample or the antibody's performance may be compromised.

1. After cell lysis and sonication, prepare Protein G magnetic beads (Table of Materials) for ChIP, mix magnetic beads until homogenous, then add 25 µL of magnetic beads to 2 mL protein low bind tubes.
   NOTE: The type of magnetic beads used will depend on the species of the antibodies.
2. Wash beads with 1 mL of blocking solution (Table 1), mix well, then place on a magnetic rack for 1 min. While still on a magnetic rack, remove supernatant once it is clear.
3. Add 1 mL of blocking solution to the magnetic beads. Rock tubes for 10 min at 4 °C on a rocking platform. Briefly spin, then place tubes on a magnetic rack and remove supernatant.
4. Repeat step 5.3 two more times.
5. Add 500 µL of blocking solution to magnetic beads. Briefly spin the antibody against Isl1 (0.04 µg/µL, Table of Materials), then add 4 µg of antibody to corresponding 2 mL protein low bind tubes containing the magnetic beads in blocking solution.
6. Repeat step 5.5 without antibody (i.e., the no antibody control for ChIP).
   NOTE: The amount of antibody to add can be determined empirically by considering the quality of the antibody and the number of cells used for ChIP.
7. Rock samples at 4 °C for 6−24 h on a rocking platform.

6. Chromatin immunoprecipitation (ChIP)

1. Wash antibody-coated beads from step 5.8 with 1 mL of blocking solution. Place samples on a rocking platform at 4 °C for 5 min. Remove supernatant.
2. Repeat step 6.1 two more times.
3. Resuspend antibody-coated beads in 50 µL of blocking solution, then transfer to new 2 mL protein low bind tubes. For each ChIP sample, add sonicated lysates (~1.0 mL from step 3.6) to antibody-coated beads in 2 mL protein low bind tubes.
4. Incubate each sample on a rocking platform overnight at 4 °C.

7. ChIP washes

NOTE: Keep samples on ice or at 4 °C to maintain protein-DNA crosslinking during ChIP washes.

1. Make high salt wash buffer, LiCl wash buffer and 10 mM Tris-HCl buffer (pH 7.4) as described in Table 2. Store in 50 mL tubes at 4 °C. Add 50 µL of 1000x CPI stock to all buffers just prior to use.
2. Briefly spin samples in 2 mL protein low bind tubes to collect liquid from the caps, then place on a magnetic rack for 1 min and remove supernatant carefully with a pipette.
3. For ChIP washes, add 1 mL of lysis buffer 3 (at 4 °C) to each tube. Mix on a rocking platform at 4 °C for 5 min. Briefly spin samples, then place on a magnetic rack for 1 minute and remove the supernatant with a pipette.
4. Add 1 mL of cold high-salt wash buffer to each tube. Mix on a rocking platform at 4 °C for 5 min. Briefly spin samples, then place on a magnetic rack for 1 min and remove the supernatant with a pipette.
5. Add 1 mL of cold LiCl wash buffer to each tube. Mix on a rocking platform at 4 °C for 5 min. Briefly spin samples, then place on a magnetic rack for 1 min and remove the supernatant with a pipette.
6. Add 1 mL of cold 10 mM Tris-HCl buffer (pH 7.4) to each tube. Mix on a rocking platform at 4 °C for 5 min. Briefly spin samples, then place on a magnetic rack for 1 min and remove the supernatant with a pipette.
   NOTE: Tris-EDTA buffer (pH 8.0) can be used instead of Tris-HCl buffer (pH 7.4).

Table 1: Recipes for lysis buffers 1−3 and blocking buffer. Store in 50 mL tubes at 4 °C. Add 50 µL of 1000x CPI (complete protease inhibitor) stock to all buffers just prior to use.

Lysis buffer 1
Reagent	Volume (mL)	[Final]
1 M HEPES (pH 7.3)	2.5	50 mM
5 M NaCl	1.4	140 mM
0.5 M EDTA (pH 8.0)	0.1	1 mM
50% Glycerol	10	10%
10% Octylphenol ethoxylate	2.5	0.50%
10% 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol	1.25	0.25%
Autoclaved ddH2O	Fill to 50
Lysis buffer 2
Reagent	Volume (mL)	[Final]
0.5 M Tris-HCl (pH 8.0)	1	10 mM
5 M NaCl	2	200 mM
0.5 M EDTA (pH 8.0)	0.1	1 mM
Autoclaved ddH2O	Fill to 50
Lysis buffer 3
Reagent	Volume (mL)	[Final]
1 M Tris-HCl (pH 8.0)	0.5	10 mM
5 M NaCl	1	100 mM
0.5 M EDTA (pH 8.0)	0.1	1 mM
10% Deoxycholic Acid	0.5	0.10%
30% N-Lauroylsarcosine sodium salt solution	0.83	0.50%
Autoclaved ddH2O	Fill to 50
Blocking solution
Reagent	Amount	[Final]
Bovine serum albumin (BSA)	250 mg	0.50%
Complete Protease Inhibitor (CPI, 1000x)	50 µL	1x
Phosphate buffered saline (PBS)	Fill to 50 mL

Table 2: Recipes for ChIP washes: High Salt Wash buffer, LiCl Wash buffer and 10 mM Tris-HCl buffer (pH 7.4). Store in 50 mL tubes at 4 °C. Add 50 µL of 1000x CPI stock to all buffers just prior to use.

High salt wash buffer
Reagent	Volume (mL)	[Final]
1 M HEPES (pH 7.3)	2.5	50 mM
5 M NaCl	5	500 mM
0.5 M EDTA (pH 8.0)	0.1	1 mM
10% 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol	5	1%
5% Deoxycholic acid	1	0.10%
5% SDS	1	0.10%
Autoclaved ddH2O	Fill to 50
LiCl wash buffer
Reagent	Volume (mL)	[Final]
0.5 M Tris-HCl (pH 8.0)	2	20 mM
0.5 M EDTA (pH 8.0)	0.1	1 mM
1 M LiCl	12.5	250 mM
10% Octylphenol ethoxylate	2.5	0.50%
5% Deoxycholic acid	5	0.50%
Autoclaved ddH2O	Fill to 50
10 mM Tris-HCl buffer
Reagent	Volume (mL)	[Final]
1 M Tris-HCl (pH 7.4)	0.5	10 mM
Autoclaved ddH2O	Fill to 50

Materialien

Name	Company	Catalog Number	Comments
Agarose, UltraPure	Invitrogen	16500	Checking Sonication (Section 4.3.6)
Albumin, Bovine Serum (BSA), Protease Free, Heat Shock Isolation, Min. 98%	BioShop	ALB003	Blocking Solution
Antibody against Isl1	DSHB	39.3F7	Antibody incuation with beads (Section 5.6)
Bioruptor Pico	Diagenode	B01060010	Sonicating Chromatin (Section 3.3)
Centrifuge 5424 R	Eppendorf	5404000138	Sonicating Chromatin (Section 3.5), Checking Sonication (Section 4.3.1, 4.3.3 and 4.3.4)
Centrifuge 5804 R	Eppendorf	22623508	Harvest, cross-linking and freezing cells (Section 1.3), Cell lysis (Section 2.2 and 2.3), Sonicating Chromatin (Section 3.1)
Complete, Mini, EDTA-free Protease Inhibitor Cocktail	Roche	4693159001	Added to all buffers, except Proteinase K buffer and ChIP Elution buffer
Deoxycholic Acid Sodium Salt	fisher scientific	BP349	Lysis Buffer 3, High Salt Wash Buffer and LiCl Wash Buffer
EDTA, 0.5 M, Sterile Solution, pH 8.0	BioShop	EDT111	Lysis Buffer 1-3, Checking Sonication (Section 4.1), High Salt Wash Buffer, LiCl Wash Buffer and ChIP Elution Buffer
Ethyl Alcohol Anhydrous, 100%	Commercial alcohols	P006EAAN	Checking Sonication (Section 4.3.3)
Formaldehyde, 36.5-38%, contains 10-15% methanol	Sigma	F8775	Harvest, cross-linking and freezing cells (Section 1.1)
Glycerol, Reagent Grade, min 99.5%	BioShop	GLY002	Lysis Buffer 1
Glycine, Biotechnology Grade, min. 99%	BioShop	GLN001	Harvest, cross-linking and freezing cells (Section 1.2)
Glycogen, RNA Grade	Thermo Scientific	R0551	Checking Sonication (Section 4.3.3)
HEPES, 1 M Sterile-filtered Solution, pH 7.3	BioShop	HEP003	Lysis Buffer 1, High Salt Wash Buffer
Lithium Chloride (LiCl), Reagent grade	Bioshop	LIT704	LiCl Wash Buffer
Magnetic beads for ChIP (Dynabeads Protein G)	Dynabeads Protein G (magnetic beads for ChIP)	Dynabeads Protein G (magnetic beads for ChIP)	Antibody incubation with beads (Section 5)
Magnetic rack (DynaMag-2 Magnet)	Invitrogen	12321D	Used in many steps in Sections: 5
N-Lauroylsarcosine sodium salt solution, 30% aqueous solution, ≥97.0% (HPLC)	Sigma	61747	Lysis Buffer 3
Octylphenol Ethoxylate (IGEPAL CA630)	BioShop	NON999	Lysis Buffer 1 and LiCl Wash Buffer
Phenol:Chloroform:Isoamyl Alcohol, Biotechnology Grade (25:24:1)	BioShop	PHE512	Checking Sonication (Section 4.3.1)
Phosphate-Buffered Saline (PBS), 1x	Corning	21040CV	Harvest, cross-linking and freezing cells (Section 1.3) and Sonicating Chromatin (Section 3.1), Antibody incuation with beads (Section 5.1)
PowerPac Basic Power Supply	BioRad	1645050	Checking Sonication (Section 4.3.6)
Proteinase K Solution, RNA Grade	Invitrogen	25530049	Checking Sonication (Section 4.2)
Rnase A, Dnase and Protease-free	Thermo Scientific	EN0531	Checking Sonication (Section 4.3.2)
Sodium chloride (NaCl), BioReagent	Sigma	S5886	Lysis Buffer 1-3, High Salt Wash Buffer
Sodium Dodecyl Sulfate (SDS), Electrophoresis Grade	BioShop	SDS001	Checking Sonication (Section 4.1), High Salt Wash Buffer and ChIP Elution Buffer
Sonication beads and 15 mL Bioruptor Tubes	Diagenode	C01020031	Sonicating Chromatin (Section 3.1 and 3.2)
ThermoMixer F1.5	Eppendorf	5384000020	Section 4.2, 4.3.2, and 4.3.5
Trizma hydrochloride solution (Tris-HCl), BioPerformance Certified, 1 M, pH 7.4	Sigma	T2194	10 mM Tris-HCl Buffer
Trizma hydrochloride solution (Tris-HCl), BioPerformance Certified, 1 M, pH 8.0	Sigma	T2694	Lysis Buffer 2, Lysis Buffer 3, Checking Sonication (Section 4.1), LiCl Wash Buffer and ChIP Elution Buffer
VWR Mini Tube Rocker, Variable Speed	VWR	10159-752	Used in many steps in sections: 1, 2, 5, 6 and 7
2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol, Triton X-100, Reagent Grade	BioShop	TRX506	Lysis Buffer 1, Sonicating Chromatin (Section 3.4) and High Salt Wash Buffer