Native Chromatin Immunoprecipitation of Neurosphere Cells

Samenvatting

Source: Mendez, F. M. et al., Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres. J. Vis. Exp. (2018).

This video demonstrates the isolation of protein-DNA complexes from neurosphere cells derived from a mouse brain tumor using native chromatin immunoprecipitation. It details the steps for chromatin preparation, immunoprecipitation, washing, and protein digestion to release DNA for downstream analysis.

Protocol

1. Native chromatin immunoprecipitation (ChIP)

1. Chromatin preparation
   1. After making stock of derived neurosphere cells (NS), culture NS in a T-75 flask in 15 mL of NSC medium at 37 °C in a tissue culture incubator with an atmosphere of 95% air and 5% CO₂ until NS become confluent. One confluent T-75 plate will yield 3-5 X 10⁶ cells.
   2. When the cells become confluent, centrifuge NS at 300 x g for 5 min, decant the supernatant, and add 1 mL of cell dissociation medium. Incubate the cells at 37 °C for 5 min. Add 5 mL of medium and count the cells using a hemocytometer.
      NOTE: 1 x 10⁶ cells are needed per immunoprecipitation (IP) reaction. Note that a pre-immune serum or IgG control must also be included.
   3. Centrifuge the NS in one 1.5 mL microcentrifuge tube at 300 x g for 5 min, decant the supernatant and resuspend NS pellet with 1 mL of balanced salt solution and transfer the suspension to low protein binding 1.5 mL microcentrifuge tubes.
   4. Centrifuge NS at 300 x g for 5 min, decant the supernatant and re-suspend the cell pellet in 95 µL of digestion buffer (50 mM Tris-HCl, pH 8.0, 1 mM CaCl₂, 0.2% polyethylene glycol octylphenyl ether) per 1 x 10⁶ cells supplemented with protease inhibitor cocktail at a high concentration (1:100). Immediately pipet the cells up and down to prevent clumping and avoid creating any bubbles.
   5. Resuspend micrococcal nuclease (MNase) in 50% glycerol to make 1 mg/mL solution (1.15 units/µL, stored at -20 °C) that will be referred to as "1x" stock. Make a "0.1x" stock by doing a second 1:9 dilution with 50% glycerol (e.g., 900 µL of "1x" MNase and 100 µL of 50% glycerol) and store it at -20 °C.
   6. Mix 5 µL of "0.1x" MNase and 145 µL of digestion buffer. Keep the enzyme on ice all the time. Add 5 µL of diluted MNase in digestion buffer per 1 x 10⁶ cells. Flick the tube to mix and place the tube in 37 °C block for exactly 12 min. Process the samples one at a time to ensure an accurate incubation time of 12 min.
   7. Add 10 µL of 10x MNase Stop Buffer (110 mM Tris-HCl, pH 8.0, 55 mM Ethylenediaminetetraacetic acid (EDTA)) per 1 x 10⁶ cells. Samples must be kept on ice from this point onwards.
   8. Add 110 µL "2x Radioimmunoprecipitation Assay buffer (RIPA) buffer" (280 mM NaCl, 1.8% polyethylene glycol octylphenyl ether, 0.2% sodium dodecyl sulfate (SDS), 0.2% Na-deoxycholate, 5 mM ethylene glycol-bis (β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA); stored at 4 °C) supplemented with protease inhibitor cocktail at high concentration (1:100) per 1 x 10⁶ cells. Flick the tube to mix.
   9. Centrifuge the tubes for 15 min in a microfuge at 4 °C and 1700 x g. Transfer the supernatant to a new regular 1.5 mL microcentrifuge tube (low protein binding microcentrifuge tubes are no longer necessary) and store them on ice. Discard the pellet.
      
2. Immunoprecipitation (IP)
   1. Mix Protein A and Protein G magnetic beads in a 1:1 ratio. For every IP, prepare 25 µL of beads.
   2. Wash the beads by adding 1 mL of RIPA buffer (10 mM Tris pH 8.0, 1 mM EDTA, 140 mM NaCl, 1% polyethylene glycol octylphenyl ether, 0.1% SDS, 0.1% Na-deoxycholate; stored at 4 °C) supplemented with protease inhibitors at low concentration (1:1000).
   3. Use a magnet to allow beads to separate from the washing solution and decant the washing solution (approximately 1 min). Perform the wash step twice.
   4. Resuspend the beads to the original volume using RIPA buffer supplemented with protease inhibitors (1:1000).
   5. If necessary, dilute the chromatin using RIPA buffer supplemented with protease inhibitors (1:1000) so that each IP has a volume of 100-200 µL. Reserve 10% of the volume used per IP of chromatin for the input.
      NOTE: For example, if 200 µL are used per IP, 20 µL of diluted chromatin should be reserved for the input. For a total of four IPs, the total chromatin volume should be diluted to 820 µL.
   6. Prepare the input. Add 100 µL of TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA) supplemented with proteinase K (0.5 mg/mL) to the input and incubate the input at 55 °C for 1 h.
   7. Purify the input using a polymerase chain reaction (PCR) purification kit following the manufacturer's instructions and elute in 50 µL of elution buffer provided in the kit.
   8. Measure the concentration of the input using a microvolume spectrophotometer and record the results in a notebook. Blank with elution buffer from kit.
      NOTE: In mouse neurospheres, we used approximately 6 µg of DNA for each IP.
   9. Run input on a 1% gel or load 1 µL on a DNA Bioanalyzer using standard settings. Store the remainder for use as input in downstream applications.
   10. Add 10 µL of washed protein A & G magnetic beads for every IP and incubate IP for 1 h at 4 °C.
       NOTE: For example, add 30 µL of protein A & G magnetic beads for three IPs.
   11. Place the samples on a magnet and allow beads to separate. Divide chromatin by transferring the previously determined amount into a new 1.5 mL tube.
   12. Add the antibody and wrap the caps with plastic paraffin film to avoid evaporation. Incubate the samples overnight at 4 °C with rotation at 20 rpm.
       NOTE: Concentration should be determined empirically following manufacturer recommendations.
   13. The next day, spin the tubes using a mini centrifuge at room temperature, 2000 x g, for 10 s. Then, add 10 µL of beads to each IP and incubate IP for 3 h at 4 °C with rotation at 20 rpm.
       
3. IP Washes and Protein Digestion
   1. Add 150 µL of RIPA buffer supplemented with protease inhibitors at low concentration (1:1000) to each IP and incubate IP at 4 °C with rotation at 20 rpm for 5 min. Place the sample on a magnetic stand and allow the magnetic beads to separate. Use a pipet to remove the supernatant. Repeat this step five times.
   2. Add 150 µL of lithium chloride (LiCl) buffer (250 mM LiCl, 10 mM Tris pH 8.0, 1 mM EDTA, 0.5% NP-40, 0.5 % Na-deoxycholate; store at 4 °C) supplemented with protease inhibitors at low concentration (1:1000) to each IP and incubate IP at 4 °C with rotation at 20 rpm for 5 min. Place the sample on a magnetic stand and allow magnetic beads to separate. Use a pipet to remove the supernatant.
   3. Add 150 µL of cold Tris-EDTA (TE) (no protease inhibitors) and incubate the sample at 4 °C with rotation at 20 rpm for 5 min. Place the sample on a magnetic stand and allow magnetic beads to separate. Use a pipet to remove the supernatant.
   4. After the final washing step, resuspend the sample in 100 µL of TE buffer supplemented with proteinase K (0.5 mg/mL) and incubate the sample at 55 °C for 1 h.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Agilent 2100 Bioanalyzer	Agilent	G2946-90004	bioanalyzer
AccuSpin Micro 17R, refrigerated	Fisher Scientific	13-100-676
C57BL/6	Taconic	B6-f	C57BL/6 mouse
Calcium Chloride	Aldrich	22350-6	buffer reagent
D-Luciferin, Potassium Salt	Goldbio	LuckK-1g
DiaMag1.5 magnetic rack	Diagenode	B04000003	for magnetic bead washes
DiaMag Rotator EU	Diagenode	B05000001	rotator
DMEM/F-12	Gibco	11330-057	NSC component
Dynabeads Protein A	Thermo Fisher Scientific	10001D	protein A magnetic beads
Dynabeads Protein G	Thermo Fisher Scientific	10003D	protein G magnetic beads
EGF	PeproTech	AF-100-15	prepare 20 μg/mL stock in 0.1% BSA and aliquot.
Ethylenediaminetetraacetic acid (EDTA)	Sigma	E-4884	buffer reagent
Ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid) (EGTA)	Sigma	E-4378	buffer reagent
Fetal Bovine Serum	Gibco	10437028	for freezing cells
FGF	PeproTech	100-18b	Prepare 20 μg/mL stock in 0.1% BSA and aliquot.
Forceps	Fine Science Tools	11008-13	for dissection of tumor
Glycerol, MB Grade	EMD- Millipore	356352
HBSS	Gibco	14175-103	balanced salt solution
Fluriso	VETone	501017	inhalation anesthetic
Ivis Spectrum	Perkin-Elmer	124262	in vivo optical imaging system
Hyqtase	HyClon	SV3003001	cell detachment media
Lithium Chloride	Sigma	L8895	buffer reagent
Low binding microtubes	Corning Costar	CLS3207	low protein binding microcentrifuge tube
Microcentrifuge tube	Fisher	21-402-903	regular microcentrifuge tube
Micrococcal Nuclease	Thermo Fisher Scientific, Affymetrix	70196Y	each batch may differ; purchase sufficient amount for experiments and aliquot.
N2	Gibco	17502-048	NSC component
Normocin	Invivogen	NOL-36-063	anti-microbial agent, use at 0.1 mg/mL.
NP-40 (Igepal CA-630)	Sigma	18896-50ML	buffer reagent
Kimble Kontes Pellet Pestle	Fisher Scientific	K749515-0000
Protease Inhibitor Cocktail	Sigma-Aldrich	P8340	aliquot and store at -20 °C.
Protinase K	Sigma-Aldrich	P2308	make 10 mg/mL stock in water; aliquot and store at -20 °C.
QIAquick PCR Purification Kit	Qiagen	28104	DNA purification kit
Scalpel	Fine Science Tools	10007-16	for dissection of tumor
Sodium Chloride	VWR	0241-5KG	buffer reagent
Sodium Deoxycholate	Sigma-Aldrich	D670-25G	buffer reagent
Sodium Dodecyl sulfate (SDS)	Sigma	L-4390	buffer reagent
Tris Base	Thermo Fisher Scientific	Bp152-1	buffer reagent
Triton X-100	Thermo Fisher Scientific	BP 151-500	polyethylene glycol octylphenyl ether
Standard Mini Centrifuge	Fisherbrand	12-006-901	standard mini centrifuge
SZX16 microscope	Olympus	SZX16	flourescent dissecting microscope
Nanodrop One	Thermo-Fisher Scientific	ND-ONEC-W