REVISED CHROMATIN IP PROTOCOL - Fred Hutch
REVISED CHROMATIN IP PROTOCOL
In obscene detail with notes for beginners
PRELIMINARY NOTES ABOUT GROWING UP CELLS:
1) For experiments with MyoD-ER, when you plate cells for experiments, plate them in growth media WITHOUT phenol red; it activates ER. Also, don’t forget to add L-Glutamine- the DME w/o phenol red lacks it.
2) For fibroblasts, grow till 80-90% confluent, then ONE MORE DAY before inducing.
3) Two plates per condition are better than 1, but count on 1 x 15cm dish per condition at least.
4) If you plan to induce cells with beta-estradiol for 2 days, make sure you have at least 3 plates per condition. The cell # from a plate induced for 2 days is about 1/2 - 1/3 of that from a 0h plate.
5) Induce the cells with phenol red free media +/- beta-estradiol, so that all the plates are treated the same. If you induce the cells with phenol read containing media, you might get some background for the 0h samples.
WHEN YOU’RE READY TO HARVESTCELLS: BY SCRAPING
1 Warm DM, DME+2%HS+insulin+transferrin (25 mL per condition), PBS
• Prepare formaldehyde fix: Need 2.5 mL per condition
For 5 mL, it should be 3.5 mL Fixation buffer
1.5 mL 37% Formaldehyde
SCRAPING PROTOCOL…
1) For the 0h high serum plates, wash the plate once with serum-free media, and add 25mL of warm, DM with insulin and transferrin but no beta-estradiol to each plate.
2) Dump the media from the cells from ONE CONDITION into a beaker and KEEP this media.
3) Take out sample for RNA (optional) - To keep some lysate for eventual RNA analysis, scrape approximately 2 squares from the 15cm plate grid of cells in remaining media (usually works out to be about 0.5mL), rinse off the cells with the remaining media in the plate, and collect the cells in a clean tube. Pour the media back into the same plate. (spin down pellet, decant liquid, store at -80ºC in step (4))
4) Add fix (2.5mL to remaining 25mL media/cells), mix by shaking ~10 times back and forth, and INCUBATE on bench for 30’, RT (try to fix the cells for the same length). If working with lots of samples, such that it takes several minutes to process all the plates, do one sample at a time from quench to fix, note the time lag between samples, and later, quench the fix at the same amount of time lag for the sample. *While cells are crosslinking, spin out cells in microfuge tube, decant sup, save pellets at –80ºC for eventual RNA preparation.
5) Quench fix with 1.4mL of 2.5M Glycine per 25mL media in a plate. Let sit for minimal 3mins (could be longer).
*NB: From this point on, try to minimize unnecessary contact between cell mix/chromatin and the container, as it sticks like no one’s business and you’ll only lose your sample. Use low bind tips, if available, and other such measures.
6) Wash the plates with 25ml PBS in the cold room.
7) Wash with 25ml CELL WASH #2, let stand in wash for minimal 10min, discard the wash in a waste beaker.
8) Wash with 25ml CELL WASH #3, let stand in wash for minimal 10min, discard the wash in a waste beaker.
9) Add 10ml of CELL WASH #3 to each 15cm plate, scrape the cells with a cell scraper, and collect the cells into 50ml tubes. Spin at 4°C, 1200rpm for 10mins, remove the supernatant until there is about 0.5-1ml buffer left in the tube.
10) Pipette the pellet into ependorf tubes.
11) Spin at 4°C, max speed for 20sec to bring down the cells. Remove the supernatant with a pipette tip.
12) For a 15cm plate, lyse pellet in 200ul Dilution/Lysis buffer/ containing protease inhibitors, and if you’re looking at histone modifications, butyrate, too. Resuspend well by pipetting up and down for 10-15 times to remove big chunks. Put on ice.
13) Sonicate each sample: max, constant, 6x 20sec, bath, setting 5. Put the tube right in the middle of the water bath to sonicate (Make sure there is always some ice in the water bath to keep the tubes cool, also you can do two tubes at the same time. The maximal volume in a tube should be ≤300ul).
14) Spin out debris from samples: 10min, 14,000 RPM, 4ºC. (You will notice there is a lot of debris, and it is OK). Transfer the soluble supernatant into an ependoff tube.
MEANWHILE….
15) Prepare 75-80ul dynabeads for each sample of 200-300ul: wash dynabeads 3× with 1ml of TE buffer for pre-clearing, resuspend, let sit on ice 3-5’, collect beads, change wash (Note: the dynabeads are hard to stick to the wall of the tube without detergent, so make sure that you use pipette tips to remove the sup, instead of sucking it out). After last wash, resuspend and bring up to 75ul in dilution/lysis buffer.
NOTE: For F5D ChIPs, Bennett uses Goat-anti-mouse IgG dynabeads,
For MyoD ChIPs, Bennett uses Protein A dynabeads.
For other Rabbit antibodies, use Protein A dynabeads.
16) Add the above precleared beads to the soluble lysates, mix by invert a few times, and keep cold on ice.
17) Bring down the dynabeads, and remove 20ul of the supernatant for INPUT. Rock the rest 180ul lysates with the precleared beads at 4ºC for at least 45mins.
18) Dilute the INPUT 1:10 in Dilution/Lysis buffer, so the final volume is 200ul. Take 2ul of the diluted INPUT for BCA assay. Store the rest INPUT at –20ºC.
BCA ASSAY- As per Pierce protocol.
a). BCA stock: 0.125ug/ul
b). BCA standards: 0ug, 0.5ug (4ul), 1ug (8ul), 2ug (16ul), 4ug (32ul).
c). 5ul of sample/standard + 1ml BCA reagent, incubate at 42ºC for 15mins, and put on ice until reading.
d). Since we use 5ul of the 1:10 dil samples, so the actual dilution factor is 1:2. (From total 200ul of cell lysates, you should get around 0.75mg~1mg protein).
STOP HERE IF YOU WANT TO - FREEZE THE SAMPLES AT -80ºC. (Bennett thinks he gets better yields if he proceeds to IP in one shot, but you may stop here, and you won’t die from it). If you do want to freeze down your samples, do it with liquid nitrogen.
19) Finally….SET UP IP’s.
a) Use 500ug (bare minimum, say your prayers, normally ~750 ug is good)-1000ug/ IP.
b) Magnet out the beads, keep the supernatant for O/N IP reactions.
c) Add 10ul (MyoD 6975B with 50% glycerol) antibody, O/N, 4ºC. Nutating.
Add 5ul (MGN F5D with no glycerol) antibody, O/N, 4ºC. Nutating.
Add 5ul (MGN SC-576 with no glycerol) antibody, O/N, 4ºC. Nutating.
Add 5ul (Acetyl H3 with no glycerol) antibody, O/N, 4ºC. Nutating.
NOTE:
a) MyoD polyclonal (6975B) and MGN F5D likes to have 0.05% SDS (if you think you are doing MyoD or MGN ChIPs, just prepare the Dilution/Lysis buffer with 0.05% SDS. Also add 0.05% SDS to subsequent IP buffers, until IP buffer 3 for best results.
b) Bennett said H3, H4, SWI/SNF antibodies do not like to have 0.05% SDS, but if you include 0.05% SDS in the buffers, the enrichment might be lowered by 2 fold or so. So not so horrible.
NEXT DAY……
20) Prepare 30ul Protein A dynabeads/IP; or 60ul IgG dynabeads/IP. Wash the bead 3 × with 1mL TE, bring up in Dilution buffer.
21) Add BSA (NEB restriction enzyme BSA) (1:10) and herring sperm/calf thymus DNA ((1:50) to dynabead blocking solution. Incubate at 4ºC, nutating, 10’.
22) Add beads to each IP reaction, incubate 4ºC, nutating, 1 hr.
For the following washes, add 1mL, pipet to resuspend, incubate 3-5’ on ice, collect beads, change the wash. Also, once you get to TE wash #1, use aerosol tips and treat the samples like PCR samples, as far as contamination care goes.
Also, after the first TE, which will have no detergent in it, the beads are considerably less adherent to the wall of the tube, so aspirate with a pipetman to be sure that if you suck something out, you can put it back.
23) WASH BEADS: (IP Wash) #2, #2, #2, #3, #3, #3, TE, TE, TE.
When in last TE wash, transfer to a fresh tube. Collect the beads.
24) ELUTE with 2 x 250ul Elution buffer, 15’, RT, shaking.
25) Bring up the input sample volume to 500ul with Elution buffer.
26) Add 20ul of 5M NaCl to elution volume and the INPUT, mix well, and incubate at 65ºC, O/N (4-16hr.) to decrosslink.
NEXT DAY….
27) Add to each sample: 10ul 0.5M EDTA, pH 8.0
20ul 1.0M Tris, pH 6.5
5ul of Proteinase K (@50 mg/mL)
INCUBATE at 45-55ºC for 2 hrs.
28) CLEAN UP* the digestion mix by two times Phenol-chloroform extraction, or with a QIAGEN PCR Cleanup Kit. (I highly recommend Phenol-chloroform extraction. In my hands, the DNA yield from Phenol-chloroform extraction is at least 3-5 fold higher comparing to that from the QIAGEN Kit)
IF DOING PHENOL-CHLOROFORM EXTRACTION:
* Before you start, make sure you have your own stock of Phenol-chloroform. The best will be to open a brand new bottle of Phenol-chloroform, and take an aliquot from it with a Glass pipette (disposable plastic pipette will be melted by Phenol-chloroform, but plastic tips are OK to use), as your own stock.
* Protocol:
1) Add equal volume of Phenol-chloroform to your DNA → vortex → spin at maximal speed at RT for 5 mins → remove the top layer (i.e., the water layer) into a new tube → discard the bottom layer (i.e., the phenol layer).
2) Do one time chloroform extraction (the steps is the same as in 1), except you add chloroform instead of Phenol-chloroform). This step removes the trace amount of Phenol in your samples.
3) ETOH precipitation (for 550ul DNA solution):
Add (1) 1/10 volumn 3M NaOAC – about 60ul.
(2) 2 volumn 100% cold ETOH – about 1.2ml.
(3) 2ul Glycogen
→ Incubate at –20ºC or –80ºC O/N
→ Spin at maximal speed at 4ºC for 20mins
→ Wash once with room temperature 70% ETOH (1ml), spin at max for 10mins.
→ Dry the pellet for 5-10mins.
4) Resuspend the pellet in 40-50ul of ChIP RESUSPENSION BUFFER (3mM Tris, pH 8.0, 0.1mM EDTA).
IF USING QIAGEN PCR Cleanup Kit:
*Add the reaction volume to 5X the volume of Qiagen Buffer PB, mix well, and purify on a PCR purification column. Wash as directed by kit. Elute in 40ul-50ul of ChIP RESUSPENSION BUFFER (3mM Tris, pH 8.0, 0.1mM EDTA).
29) Take the OD of input samples, and get ready to set up the PCR’s. Store samples at –20ºC when not in use. *You may not have enough IP sample to detect an accurate OD. I usually start my ChIP PCR’s with 3-4.5ul of my elution per reaction and see how it goes.
Bennett: A typical PCR reaction (for sybergold):
15 ul/ rxn 20 ul/ rxn 15rxn 20rxn 25rxn 30rxn 35rxn 40rxn
IP sample 3 ul 3 ul
H2O 0.5 ul 1.5 ul
dNTP(10mM)+buffer(1:1) 3 ul 4 ul 60ul 80ul 100ul 120ul 140ul 160ul
100% DMSO 0.75 ul 1 ul 15ul 20ul 25ul 30ul 35ul 40ul
Primer A (10uM) 1.5 ul 2 ul 30ul 40ul 50ul 60ul 70ul 80ul
Primer B (10uM) 1.5 ul 2 ul 30ul 40ul 50ul 60ul 70ul 80ul
Amy primerL (10uM) 1.5 ul 2 ul 15.5ul 30ul 40ul 50ul 60ul 70ul 80ul
Amy primerR (10uM) 1.5 ul 2 ul 30ul 40ul 50ul 60ul 70ul 80ul
Mg++ (50mM) 1 ul 1.5 ul 22.5ul 30ul 37.5ul 45ul 52.5ul 60ul
15% Triton 0.4 ul 0.5 ul 7.5ul 10ul 12.5ul 15ul 17.5ul 20ul
Platinum Taq 0.35 ul 0.5 ul 7.5ul 10ul 12.5ul 15ul 17.5ul 20ul
Total volumn 15 ul 20 ul
Quadri-plex PCR(HIS) 20 ul/ rxn 5rxn 15rxn 20rxn 25rxn 30rxn 35rxn 40rxn
IP sample 1 ul
H2O 1.5 ul 7.5ul 22.5ul 30ul 37.5ul 45ul 52.5ul 60ul
dNTP(10mM)+buffer(1:1) 4 ul 20ul 60ul 80ul 100ul 120ul 140ul 160ul
100% DMSO 1 ul 5ul 15ul 20ul 25ul 30ul 35ul 40ul
Primer A1 (20uM) 1 ul 5ul 15ul 20ul 25ul 30ul 35ul 40ul
Primer A2 (20uM) 1 ul 5ul 15ul 20ul 25ul 30ul 35ul 40ul
Primer B1 (20uM) 1 ul 5ul 15ul 20ul 25ul 30ul 35ul 40ul
Primer B2 (20uM) 1 ul 17.5 5ul 15ul 20ul 25ul 30ul 35ul 40ul
Primer C1 (20uM) 1 ul 5ul 15ul 20ul 25ul 30ul 35ul 40ul
Primer C2 (20uM) 2 ul 5ul 15ul 20ul 25ul 30ul 35ul 40ul
Amy primerL (20uM) 2 ul 10ul 30ul 40ul 50ul 60ul 70ul 80ul
Amy primerR (20uM) 1 ul 10ul 30ul 40ul 50ul 60ul 70ul 80ul
Mg++ (50mM) 1.5 ul 7.5ul 22.5ul 30ul 37.5ul 45ul 52.5ul 60ul
15% Triton 0.5 ul 2.5ul 7.5ul 10ul 12.5ul 15ul 17.5ul 20ul
Platinum Taq 0.5 ul 2.5ul 7.5ul 10ul 12.5ul 15ul 17.5ul 20ul
Total volumn 20 ul
Quadri-plex PCR(MyoD) 20 ul/ rxn 5rxn 15rxn 20rxn 25rxn 30rxn 35rxn 40rxn
IP sample 5 ul
dNTP(10mM)+buffer(1:1) 4 ul 20ul 60ul 80ul 100ul 120ul 140ul 160ul
100% DMSO 1 ul 5ul 15ul 20ul 25ul 30ul 35ul 40ul
Primer A1 (20uM) 1 ul 5ul 15ul 20ul 25ul 30ul 35ul 40ul
Primer A2 (20uM) 1 ul 5ul 15ul 20ul 25ul 30ul 35ul 40ul
Primer B1 (20uM) 1 ul 5ul 15ul 20ul 25ul 30ul 35ul 40ul
Primer B2 (20uM) 1 ul 15.1 5ul 15ul 20ul 25ul 30ul 35ul 40ul
Primer C1 (20uM) 1 ul 5ul 15ul 20ul 25ul 30ul 35ul 40ul
Primer C2 (20uM) 1 ul 5ul 15ul 20ul 25ul 30ul 35ul 40ul
Amy primerL (50uM) 0.8 ul 4ul 12ul 16ul 20ul 24ul 28ul 32ul
Amy primerR (50uM) 0.8 ul 4ul 12ul 16ul 20ul 24ul 28ul 32ul
Mg++ (50mM) 1.5 ul 7.5ul 22.5ul 30ul 37.5ul 45ul 52.5ul 60ul
15% Triton 0.5 ul 2.5ul 7.5ul 10ul 12.5ul 15ul 17.5ul 20ul
Platinum Taq 0.5 ul 2.5ul 7.5ul 10ul 12.5ul 15ul 17.5ul 20ul
Total volumn 20 ul
94°C 2min
94°C 45sec
62°C 1min (30-32)×
68°C 1.5min
68°C 7min
4°C O/N
Duplex PCR(MyoD) 20 ul/ rxn 5rxn 15rxn 20rxn 25rxn 30rxn 35rxn 40rxn
IP sample 2 ul
H20 6.5ul 32.5ul 97.5ul 130ul 162.5 195ul 227.5 260ul
dNTP(10mM)+buffer(1:1) 4 ul 20ul 60ul 80ul 100ul 120ul 140ul 160ul
100% DMSO 1 ul 5ul 15ul 20ul 25ul 30ul 35ul 40ul
Primer A1 (20uM) 1 ul 5ul 15ul 20ul 25ul 30ul 35ul 40ul
Primer A2 (20uM) 1 ul 5ul 15ul 20ul 25ul 30ul 35ul 40ul
Primer B1 (20uM) 1 ul 5ul 15ul 20ul 25ul 30ul 35ul 40ul
Primer B2 (20uM) 1 ul 18 5ul 15ul 20ul 25ul 30ul 35ul 40ul
Mg++ (50mM) 1.5 ul 7.5ul 22.5ul 30ul 37.5ul 45ul 52.5ul 60ul
15% Triton 0.5 ul 2.5ul 7.5ul 10ul 12.5ul 15ul 17.5ul 20ul
Platinum Taq 0.5 ul 2.5ul 7.5ul 10ul 12.5ul 15ul 17.5ul 20ul
Total volumn 20 ul
Diane: Just do a regular PCR. i.e., comparing to Bennett and Miriam, she uses ¼ of the dNTP, no DMSO or Triton, ¼ of each primer, ½ of Mg++. Also she uses the following cycles:
94°C 2min
94°C 30sec
62°C 30sec (30-32)×
72°C 1min
72°C 7min
4°C O/N
Miriam: A typical PCR reaction looks like this (20ul final volume):
2ul IP sample
2ul 10x PCR buffer
2ul dNTP mix (10mM each)
2ul DMSO
0.2ul BSA (100X)
25pmol each primer per reaction
1.6ul Mg++ (50mM)
0.33ul Platinum Taq
0.1ul − dCTP
SyberGold Staining:
1) Run 3% low-melt+1%Agarose gel in TAE buffer.
Note:
a) use the tray and gel box without EtBr. A gel box with EtBr increases the background a lot.
b) For a big gel, pour 65ml of Agarose.
c) use orange sample loading dye.
d) dilute the 100bp ladder 1:20, and load 5ul/lane as the molecular marker.
e) run the gel until the orange dye just goes into the buffer.
2) Stain the gel with 1: 10,000 SyberGold for an hour (plastic container without EtBr will be the best for staining. SyberGold might stain glassware, and it is hard to remove), cover your tray with Aluminum Foil (since the SyberGold is light sensitive).
3) Bring the gel down to the Image facility, rinse the gel once with water, so there won’t be excessive SyberGold left on the scanner.
4) Scan the Gel.
*Sybergold has ‘Excitation Max’ of 495nm, and ‘Emission Max’ of 537nm.
a) choose the Acquisition Mode to be: ‘Fluorescence’.
b) Emission filter: Fluorescein-526sp, PMT-600/570, Laser-Green 532, Sensitivity-normal.
c) Focal Plane: either Platen or +3mm, or try both.
d) Scan it on the highest resolution (200 micron).
e) Take the saved ImageQuat file into Image Quat tools, choose ‘Noise filter’, choose ‘median filter’.
SOLUTIONS FOR CHROMATIN IP
STOCKS: all filtered with 0.2um filter
| |Buffer Name |MW(g/mol) |Powder weighed |Final volumn |
|1 |1M Hepes (pH=8) |26 |2.6g |100ml |
|2 |0.5M EDTA (pH=8) | |Lab stock | |
|3 |0.5M EGTA (pH=8) |380 |38g |200ml |
|4 |1M Tris (pH=8) |121 |121g |1L |
|5 |1M Tris (pH=8.1) |121 |60.5g |500ml |
|6 |1M Tris (pH=6.5) |121 |12.1g |100ml |
|7 |20% SDS | |Lab stock | |
|8 |20% Triton | |20ml |80ml |
|9 |20% NP-40 | |20ml |80ml |
|10 |20% Na-Deoxycholate | |20ml |80ml |
|11 |5M LiCl |42.4 |42.4g |200ml |
|12 |2.5 M Glycine |75 |37.5g |200ml |
|13 |5M NaCl | |Lab stock | |
|14 |0.25M sodium Butyrate |110 |2.75g |100ml |
|15 |3M Sodium Acetate |82 |24.6g |100ml |
10X FORMALDEHYDE FIXATION BUFFER
Reagent stock vol (100mL)
50mM HEPES, pH 8.0 1.0M 5mL
1mM EDTA, pH 8.0 0.5M 200ul
0.5mM EGTA 0.5M 100ul
100mM NaCl 5.0M 2mL
*Add 37% Formaldehyde FRESH before use…. 3.5 mL 10X Buffer + 1.5 mL 37% Formaldehyde
Chromatin CELL WASH #2 BUFFER (Keep at 4 degree the day before use)
reagent stock vol (100ml) vol (500ml) vol(1L)
10mM Tris, pH 8.0 1.0M 1ml 5ml 10ml
10mM EDTA, pH 8.0 0.5M 2ml 10ml 20ml
0.5mM EGTA 0.5M 100ul 500ul 1ml
0.25% Triton-X 20% 1.25ml 6.25ml 12.5ml
*Add Sodium Butyrate FRESH before use, if doing an acetylation-type IP…
10mM Butyrate 0.25M 4mL 20ml 40mL
Chromatin CELL WASH #3 BUFFER (Keep at 4 degree the day before use)
reagent stock vol (100mL) vol (500mL) vol(1L)
10mM Tris, pH 8.0 1.0M 1ml 5ml 10ml
1mM EDTA, pH 8.0 0.5M 200ul 1ml 2ml
0.5mM EGTA 0.5M 100ul 500ul 1ml
200mM NaCl 5M 4ml 20ml 40ml
*Add Sodium Butyrate, as above, if needed.
10mM Butyrate 0.25M 4mL 20ml 40mL
“DILUTION”/LYSIS BUFFER (Keep at 4 degree the day before use)
reagent stock vol (2ml) vol(10ml) vol(100ml) vol (200ml)
1.1% Triton-X 20% 111ul 555ul 5.6ml 11.2ml
4mM EDTA, pH 8.0 0.5M 16ul 80ul 800ul 1.6ml
40mM Tris, pH 8.1 1.0M 80ul 400ul 4.0ml 8.0ml
300mM NaCl 5.0M 120ul 600ul 6.0ml 12.0ml
*10mM Butyrate, if needed 0.25M 80ul 400ul 4ml 8.0ml
H2O 1.393ml 6.965ml 69.65ml 139.3ml
--------------------------------------------------------------------------------------------------------------------------
Inhibitor Cocktail 10X 200ul 1ml 10ml 20ml
(from Boehringer pellet, add right before use)
--------------------------------------------------------------------------------------------------------------------------
IP WASH BUFFER #2 (Keep at 4 degree the day before use)
reagent stock volume (100mL) volume (200mL)
1% Triton-X 20% 5mL 10mL
2mM EDTA, pH 8.0 0.5M 400ul 800ul
20mM Tris, pH 8.1 1.0M 2mL 4mL
500mM NaCl 5.0M 10mL 20mL
H20 ---- 82.6mL 165mL
**Add SDS as needed/preferred by the specific antibody (MyoD IP- add SDS to 0.05%)
IP WASH BUFFER #3 (Keep at 4 degree the day before use)
reagent stock volume (100mL) volume (200mL)
0.25M LiCl 5M 5mL 10mL
1% NP-40 20% 5mL 10mL
1% Na-Deoxycholate 20% 5mL 10mL
1mM EDTA, pH 8.0 0.5M 200ulL 400uL
10mM Tris, pH 8.1 1.0M 1mL 2mL
H20 ---- 83.8mL 167.6mL
**Add SDS as needed/preferred by the specific antibody (MyoD IP- add SDS to 0.05%)
ELUTION BUFFER (Keep at RT all the time to avoid precipitation. Bennett includes EDTA in his ELUTION BUFFER, while Diane does not. Your call.)
Reagent stock volume (100mL)
1% SDS 20% 5mL
0.1M NaHCO3 (1mol=84.01g) ---- 0.84g
10mM EDTA 0.5M 1ml
ad 100 mL with H20
ChIP RESUSPENSION BUFFER
reagent stock volume (50mL)
3mM Tris, pH=8 1M 150uL
0.1mM EDTA 0.5M 10uL
TE buffer
Reagent stock volume (500mL)
10mM Tris, pH=8 1M 5mL
1mM EDTA 0.5M 1ml
ad 500 mL with H20
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