ChIP: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
(→Notes) |
|||
Line 90: | Line 90: | ||
# collect the beads with magnet wash beads in 1.5 ml BSA/PBS 2 times, collect the beads with the magnet. | # collect the beads with magnet wash beads in 1.5 ml BSA/PBS 2 times, collect the beads with the magnet. | ||
# Add 6-10 µg of Ab + 250µL of PBS/BSA. | # Add 6-10 µg of Ab + 250µL of PBS/BSA. | ||
# incubate 4 hr to O.N. on a rotating platform at | # incubate 4 hr to O.N. on a rotating platform at 4°C. | ||
# wash beads 3 times in 1.5 ml PBS/BSA. | # wash beads 3 times in 1.5 ml PBS/BSA. | ||
# Resuspend in 100µL PBS/BSA. | # Resuspend in 100µL PBS/BSA. | ||
Line 96: | Line 96: | ||
===III. Cell Sonication=== | ===III. Cell Sonication=== | ||
* Note: Add protease inhibitors to all lysis buffers before use. If using a protease inhibitor cocktail tablet from Complete, dissolve one tablet in 2 ml H2O for 25x solution. Use 400uL per 10mL of buffer. Store in aliquots at -20°C. | |||
# Resuspend each tube of cells in 5-10 ml of Lysis buffer I. Rock at 4°C for 10’. Then spin at 2500rpm in a table top centrifuge, 3 min at 4°C. | |||
# Resuspend each tube of cells in 5-10 ml of buffer 2. Rock gently at 4°C for 5 min. Pellet nuclei in tabletop centrifuge by spinning at 2500 rpm, 3 min at 4°C. | |||
# Resuspend pellet in each tube in 3 ml buffer 3 and put into 15mL conical tube cut off at the 6.5mL mark. | |||
# Sonicate the suspension using the automatic sonicator. | |||
#* Appropriate sonication time needs to be determined for each cell type. For example, HepG2 cells: 6 cycles of 30 seconds with 1 minute intervals at power 7.0 (~40 watts). | |||
# Add 1/10 volume of 10% Triton X-100. Transfer to 1.5 ml centrifuge tubes. Spin out debris 14K, 4°C, 10 min. | |||
# If you are using one tube of cells for more than one ChIP then you need to add more LB3 and 10% Triton X-100 so that the volume ends up being 3 mL of cells in LB3 with 300uL of Triton per ChIP. | |||
# Save 50 µL (or at least 1/50 vol) of cell lysate from each sample as input control. Store at -20°C. | |||
===IV. Chromatin Immunoprecipitation=== | ===IV. Chromatin Immunoprecipitation=== | ||
#Add 100 µL Ab prebound Dynal magnetic beads from step III. | #Add 100 µL Ab prebound Dynal magnetic beads from step III. | ||
#Rock | #Rock 4°C 6 hrs to O/N. | ||
===V. Washing, eluting, and reverse cross-linking=== | ===V. Washing, eluting, and reverse cross-linking=== | ||
* Work in the cold room until step 6. | |||
# Transfer IP reactions to centrifuge tubes | |||
# Use the magnetic stand to precipitate the beads. | |||
# Wash 4-8 times with 1 mL wash buffer (Ripa buffer) | |||
# Wash once with 1 ml TE-plus-50 mM NaCl or 1 mL TBS. | |||
# Spin 3k for 2-3 min and aspirate any residual TE/TBS. | |||
# Add 200 µl of elution buffer. | |||
# Elute DNA-protein complexes from beads at 65°C for 10-15 min with brief vortexing every 2 min. | |||
# Spin down beads 14k for 1 min. | |||
# Remove all 200 µl of supernatant. | |||
# Reverse x-link O/N 65°C. Min 6 hours | |||
# Thaw input from step III(6), add 3 vol of elution buffer and reverse x-link O/N 65°C. | |||
===VI. RNase, Proteinase K=== | ===VI. RNase, Proteinase K=== | ||
# Add 1 vol of TE to IP and input (wce) fraction. | |||
# Add RNase A so final is 0.2µg/µL (~5 µL/250 µL rxn). Incubate 37∞C 1-2hr. | |||
# Add proteinase K so final is 0.2µg/µL (~2.5 µL/250 µL rxn). Incubate 55∞C 1-2hr. | |||
# Extract once w/ 1 vol of phenol. | |||
# Extract once w/ 1 vol of phenol:chl:IA (made by mixing 1 vol of phenol w/ 1 vol of Chloroform:isoamyl alcohol). | |||
# extract once w/ 1 vol of chl:IA. | |||
#* Alternatively: extract 1x w/ 1 vol phenol:chl:IA using phaselock tubes | |||
# add 20 µg (1 µL) of glycogen. | |||
# Add 5M NaCl so final is 0.2M (10 µL/250 µL rxn). | |||
# Add 2X volume of EtOH. Precipitate DNA with a 10 min 14K spin, and wash pellet with 500 µL 75% EtOH. | |||
# Dry and resuspend pellets in 60 µL 10mM Tris HCl pH 8. | |||
# Normalize the input/wce fraction to 100 ng/µL using the Nanodrop. | |||
===VII. T4 DNA Polymerase Fill-in=== | |||
# To 60 µL of IP sample, and 200 ng (=2 µL) of the normalized input (wce) diluted to 60 µL, add: | |||
#*10 µL of 10X T4 DNA pol buffer | |||
#* 0.5 µL of BSA (NEB 10mg/ml) | |||
#* 0.4 µL of 25mM dNTP mix | |||
#* 0.2 µL of T4 DNA pol (NEB 3U/µL) | |||
#* 28.9 µL of H2O | |||
#* final volume =100uL | |||
#* Incubate 12∞C 20 min in water bath. | |||
#* Add 12 µl 3 M NaOAc and 1.0 µL glycogen. | |||
#* Extract 1x with 120 µL phenol:chl:IA. | |||
#* Precipitate with 300 µL EtOH. | |||
#* Spin and wash with 500 µL 75% EtOH. | |||
#* Dry pellet and resuspend in 25 µL H2O. | |||
===VIII. Blunt-end ligation=== | |||
# Make at 4°C, 25 µL ligase mix per reaction: | |||
#* 7.8 µL of H2O | |||
#* 10 µL of 5X ligase buffer (Gibco) | |||
#* 6.7 µL of annealed linkers (thaw at 4°C) | |||
#* 0.5 µL of T4 DNA ligase | |||
# add mix to 25 µL of sample. | |||
# incubate 12∞C O/N, cover with foil. | |||
# Next day add 6µL of 3M NaOAc and 150 µL EtOH. | |||
# Spin and wash with 500 µL 75% EtOH. | |||
# Dry and resuspend pellet in 40 µL PCR reaction mix below. | |||
''To Be Continued'' ~ [[User:Cconboy|cmc]] 18:18, 21 August 2006 (EDT) | |||
==Notes== | ==Notes== |
Revision as of 15:18, 21 August 2006
This protocol is in development.~ cmc 15:03, 21 August 2006 (EDT)
Overview
Replace this sentence with a brief description of the protocol and its goal.
Materials
11% Formaldehyde Solution
per 50 ml: (Final concentration)
- 14.9 ml of 37% Formaldehyde (11%)
- 1 ml of 5M NaCl (0.1M)
- 100 µl of 0.5 M EDTA, pH 8 (1 mM)
- 50 µl of 0.5 M EGTA, pH 8 (0.5 mM)
- 2.5 ml of 1M Hepes, pH 8 (50 mM)
2.5 M glycine (187.5 g/L)
- Dissolve glycine in water with constant stirring
- Don’t adjust pH
BSA/PBS Solution
per 100 mL:
- 10 mL of 10x PBS
- 500 mg of BSA
- 90 mL of H20
(good for at least a week)
Lysis Buffer 1 (LB1)
per 100 ml: (final concentration)
- 5 ml of 1M Hepes-KOH, pH 7.5 (50 mM)
- 2.8 ml of 5M NaCl (140 mM)
- 0.2 ml of 0.5M EDTA (1 mM)
- 10 ml of glycerol (10%)
- 5 ml of 10% NP-40 (0.5%)
- 0.25 ml of Triton X-100 (0.25%)
Lysis Buffer 2 (LB2)
per 100 ml: (final concentration)
- 4 ml of 5M NaCl (200 mM)
- 0.2 ml of 0.5M EDTA (1 mM)
- 0.1 ml of 0.5M EGTA (0.5 mM)
- 1.0 ml of 1M Tris pH 7.5 (10 mM)
Lysis Buffer 3 (LB3)
per 100 ml: (final concentration)
- 0.2 ml of 0.5M EDTA (1 mM)
- 0.1 ml of 0.5M EGTA (0.5 mM)
- 1.0 ml of 1M Tris-HCl, pH7.5 (10 mM)
- 2 mL of 5M NaCl (100 mM)
- 1 mL of 10% Na-Deoxycholate (0.1%)
- 500 mg of N-lauroyl sarcosine (0.5%)
Wash buffer (RIPA buffer)
per 100ml: (final concentration)
- 5ml of 1M Hepes, pH 7.6 (50 mM)
- 200µL of 0.5M EDTA (1 mM)
- 7 ml of 10% DOC (Na deoxycholate) (0.7%)
- 10 ml of 10% NP-40 (IPGEL) (1%)
- 10 ml of 5M LiCl or 2.12g powder (0.5 M)
Elution buffer
- 50mM Tris pH8
- 1mM EDTA
- 1% SDS
Procedure
I. Cell cross-linking
- Use 5x107 – 1x108 cells (70-80% confluency for adhesion cells of 8-12 15 cm2 plates or 175 cm2 flasks) for each ChIP reaction.
- Adherent cells:
- Add 1/10 volume of fresh 11% formaldehyde solution to plates.
- Swirl plates briefly and let them sit at RT for 10 min.
- Add 1/20 volume of 2.5 M glycine to plates to quench formaldehyde.
- Rinse cells twice with 5 ml 1X PBS. Harvest cells using silicon scraper.
- Spin cells at 4k for 10’ at 4°C.
- Transfer cells to 15ml conical tubes and spin 4k 10’ at 4°C.
- Flash freeze cells in liquid nitrogen and store pellets at –80 °C.
- Suspension cells:
- Add 1/10 volume of fresh 11% formaldehyde solution to flasks.
- Swirl flasks briefly and let them sit at RT for 20 min.
- Add 1/20 volume of 2.5 M glycine to flasks to quench formaldehyde.
- Spin cells at 2500rpm for 10’ at 4°C.
- Rinse cells with 100ml 1x PBS and spin cells at 2500rpm for 10’ at 4°C.
- Rinse cells with 10ml 1x PBS and transfer to 15ml conical tubes and spin 4k 10’ at 4°C.
- Flash freeze cells in liquid nitrogen and store pellets at –80 °C.
II. Preblock and binding of antibody to magnetic beads
- Keep beads on ice for all steps
- wash 100 µL Dynal magnetic beads (per reaction) in 1 ml fresh BSA/PBS
- collect the beads with magnet wash beads in 1.5 ml BSA/PBS 2 times, collect the beads with the magnet.
- Add 6-10 µg of Ab + 250µL of PBS/BSA.
- incubate 4 hr to O.N. on a rotating platform at 4°C.
- wash beads 3 times in 1.5 ml PBS/BSA.
- Resuspend in 100µL PBS/BSA.
III. Cell Sonication
- Note: Add protease inhibitors to all lysis buffers before use. If using a protease inhibitor cocktail tablet from Complete, dissolve one tablet in 2 ml H2O for 25x solution. Use 400uL per 10mL of buffer. Store in aliquots at -20°C.
- Resuspend each tube of cells in 5-10 ml of Lysis buffer I. Rock at 4°C for 10’. Then spin at 2500rpm in a table top centrifuge, 3 min at 4°C.
- Resuspend each tube of cells in 5-10 ml of buffer 2. Rock gently at 4°C for 5 min. Pellet nuclei in tabletop centrifuge by spinning at 2500 rpm, 3 min at 4°C.
- Resuspend pellet in each tube in 3 ml buffer 3 and put into 15mL conical tube cut off at the 6.5mL mark.
- Sonicate the suspension using the automatic sonicator.
- Appropriate sonication time needs to be determined for each cell type. For example, HepG2 cells: 6 cycles of 30 seconds with 1 minute intervals at power 7.0 (~40 watts).
- Add 1/10 volume of 10% Triton X-100. Transfer to 1.5 ml centrifuge tubes. Spin out debris 14K, 4°C, 10 min.
- If you are using one tube of cells for more than one ChIP then you need to add more LB3 and 10% Triton X-100 so that the volume ends up being 3 mL of cells in LB3 with 300uL of Triton per ChIP.
- Save 50 µL (or at least 1/50 vol) of cell lysate from each sample as input control. Store at -20°C.
IV. Chromatin Immunoprecipitation
- Add 100 µL Ab prebound Dynal magnetic beads from step III.
- Rock 4°C 6 hrs to O/N.
V. Washing, eluting, and reverse cross-linking
- Work in the cold room until step 6.
- Transfer IP reactions to centrifuge tubes
- Use the magnetic stand to precipitate the beads.
- Wash 4-8 times with 1 mL wash buffer (Ripa buffer)
- Wash once with 1 ml TE-plus-50 mM NaCl or 1 mL TBS.
- Spin 3k for 2-3 min and aspirate any residual TE/TBS.
- Add 200 µl of elution buffer.
- Elute DNA-protein complexes from beads at 65°C for 10-15 min with brief vortexing every 2 min.
- Spin down beads 14k for 1 min.
- Remove all 200 µl of supernatant.
- Reverse x-link O/N 65°C. Min 6 hours
- Thaw input from step III(6), add 3 vol of elution buffer and reverse x-link O/N 65°C.
VI. RNase, Proteinase K
- Add 1 vol of TE to IP and input (wce) fraction.
- Add RNase A so final is 0.2µg/µL (~5 µL/250 µL rxn). Incubate 37∞C 1-2hr.
- Add proteinase K so final is 0.2µg/µL (~2.5 µL/250 µL rxn). Incubate 55∞C 1-2hr.
- Extract once w/ 1 vol of phenol.
- Extract once w/ 1 vol of phenol:chl:IA (made by mixing 1 vol of phenol w/ 1 vol of Chloroform:isoamyl alcohol).
- extract once w/ 1 vol of chl:IA.
- Alternatively: extract 1x w/ 1 vol phenol:chl:IA using phaselock tubes
- add 20 µg (1 µL) of glycogen.
- Add 5M NaCl so final is 0.2M (10 µL/250 µL rxn).
- Add 2X volume of EtOH. Precipitate DNA with a 10 min 14K spin, and wash pellet with 500 µL 75% EtOH.
- Dry and resuspend pellets in 60 µL 10mM Tris HCl pH 8.
- Normalize the input/wce fraction to 100 ng/µL using the Nanodrop.
VII. T4 DNA Polymerase Fill-in
- To 60 µL of IP sample, and 200 ng (=2 µL) of the normalized input (wce) diluted to 60 µL, add:
- 10 µL of 10X T4 DNA pol buffer
- 0.5 µL of BSA (NEB 10mg/ml)
- 0.4 µL of 25mM dNTP mix
- 0.2 µL of T4 DNA pol (NEB 3U/µL)
- 28.9 µL of H2O
- final volume =100uL
- Incubate 12∞C 20 min in water bath.
- Add 12 µl 3 M NaOAc and 1.0 µL glycogen.
- Extract 1x with 120 µL phenol:chl:IA.
- Precipitate with 300 µL EtOH.
- Spin and wash with 500 µL 75% EtOH.
- Dry pellet and resuspend in 25 µL H2O.
VIII. Blunt-end ligation
- Make at 4°C, 25 µL ligase mix per reaction:
- 7.8 µL of H2O
- 10 µL of 5X ligase buffer (Gibco)
- 6.7 µL of annealed linkers (thaw at 4°C)
- 0.5 µL of T4 DNA ligase
- add mix to 25 µL of sample.
- incubate 12∞C O/N, cover with foil.
- Next day add 6µL of 3M NaOAc and 150 µL EtOH.
- Spin and wash with 500 µL 75% EtOH.
- Dry and resuspend pellet in 40 µL PCR reaction mix below.
To Be Continued ~ cmc 18:18, 21 August 2006 (EDT)
Notes
- This protocol will likely fail the first time you try it. Your LM-PCR product will contain no DNA. Don't loose heart. Try, try again. (Hard to pin down exactly what goes wrong between sonication and LM-PCR, but it seems that the blunting step is very sensitive. Most people get it to work by trying two or three times.) ~ cmc 16:25, 21 August 2006 (EDT)