染色质免疫共沉淀及3C.doc

ChIP Protocol Reagents (avoid contamination): Fix: 1% Formaldehyde in PBS. Glycine stop: 0.125 M Glycine in PBS Cell Collection: Tris-HCl 100 mM pH=9.4 DTT 10 mM Protease inhibitors (1mM PMSF; 1*cocktail)Nucleus/Chromatin Preparation (NCP). Buffer I: EDTA 10 mM Buffer II: EDTA 1 mM EGTA 0.5 mM EGTA 0.5 mM Hepes 10 mM pH=6.5 Hepes 10 mM pH=6.5 Triton X-100 0.25% NaCl 200 mM Lysis Buffer: EDTA 10 mM Tris-HCl 50 mM pH=8.1 SDS 1% IP Buffer:SDS 0.01% EDTA 2 mM NaCl 150 mM Tris-HCl 20 mM pH=8.1 Triton X-100 1% Protease inhibitors WashingsBuffer I: EDTA 2 mM Buffer II: EDTA 2 mM Tris-HCl 20 mM pH=8.1 Tris-HCl 20 mM pH=8.1 SDS 0.1% SDS 0.1 % Triton X-100 1% Triton X-100 1% NaCl 150 mM NaCl 500 mM Buffer III:EDTA 1 mM Buffer IV: EDTA 1 mM Tris-HCl 10 mM pH=8.1 Tris-HCl 10 mM pH=8.1 LiCl 250 mM Deoxycholate 1 % NP-40 1% Extraction Buffer:SDS 1% NaHCO3 0.1M Volumes are given for 5 x 106 Cells. 1- Fixing.A) Collect suspended cells and tip out one extra volume cells for estimation of cell number. Then directly fix the cells in the culture medium to a final concentration of 1% formaldehyde (For example, add 278ul 37% formaldehyde into 10ml cell solution) and incubate for 10 min at Room Temp. B) Alternatively, wash adherent cells twice with PBS. Fix the cells with 2 ml 1% formaldehyde for 10 min at Room Temp. 2- Stop Cross-linking. A) Add the Glycine to a final concentration of 0.125 M (500ul 2.5M into 10ml) and incubate 5 min at Room Temp. Wash the cells twice with 5 ml cold PBS. Collect the cells in 1 ml cold Cell Collection Buffer. Incubated on ice for 15 min and subsequently at 30C for 15 min. B) Alternatively, aspirate medium, removing as much medium as possible. Wash the cells twice with 2 ml ice cold PBS. Collect the cells in 1 ml cold Cell Collection Buffer. Chill on ice for 10 min, and 15 min at 30C. 3- Preparation of Cell nuclei/Chromatin fragmentation with SDS derived method sequentially by vortexing. Spin Cells 5 min at 2000 g .Washings/Resuspend Wash/Spin 3000 g 5 min at 4C each time1 ml of cold PBS 1 ml of NCP Buffer I/on ice 10min 1 ml of NCP Buffer II/on ice 10min Lysis: Resuspend in 350 l of Lysis Buffer, incubate on ice30min Sonicate 79 times 20 seconds at 200W output, with 1 minutes refractory period between sonications. Ensure to keep the lysate on ice during this time. 4- Remove 10 l for detecting the sonication efficiency to an average length of about 200500bp. Expanding the reaction system to final volume 50ul, then add 2l 5M NaCl and 20g ProK, and incubate this fraction at 65C overnight (reverse the cross-linking). Next day, add 10g RNaseA at 37C for another 1 hour. Isolate DNA by phenol/chloroform extraction and run sample (5l, 10l samples) in a 1.5% agarose agarose gel and visualize by Ethidium Bromide staining to detect shearing efficiency. Repeat these latter steps until the desired fragment size is obtained. 5- Once the chromatin has been appropriately sonicated, debris is cleared by centrifugation at maximum speed for 10 min at 4C. This lysate can now be stored at 4C for less than a week. Remove 1/10 as Input and froze it at -20C until cross-linking reversal step.Split cell lysate into two halves (IgG and IP). We can measure the protein concentration of the lysate and adjust the concentration to 1g/l by addition of IP buffer (add Protease inhibitors). Alternatively, we can dilute 300l lysate (5*106 cells) to 2ml. Dilute at least 6 fold (10 fold is good).6- Preclearing and reducing nonspecific background. Add: 4g IgG or 20 l preimmune serum (same origin with antibody) 35 l of the 50 % Protein A/G-agarose beads slurry (with high affinity with IgG or antibody)Incubate 2-4 hours at cold room under rocking. Pellet the unspecific complexes by centrifugation at 800 g for 5 min at 4C. 7- Immunoprecipitation.Add 4g IgG or specific antibody into two halves respectively.Incubate under rocking overnight at 4C.8- Add 35 l of the 50 % Protein A/G-agarose beads slurry into two halves respectively. Incubate under rocking for 4 hours at 4C.9- Pellet the specific complexes by centrifugation at 800 g for 3 min at 4C. Remove the supernatant as much as possible. 10- Serially wash the pellet. Wash/Spin 800 g for 3 min at 4C each time. - 900 l of Washing Buffer I (3 times) - 900 l of Washing Buffer II - 900 l of Washing Buffer III - 900 l of Washing Buffer IV 3 times 11- Extraction of precipitated chromatin complexes. Extract 2 times the specific DNA/protein complexes by adding for the first time 220 l of Extraction Buffer (freshly prepared) to the beads pellet, vortex briefly and mix occasionally for 30min rotation at room temperature; and the subsequent repeats with 220 l Extraction Buffer and 10 min incubation times. Centrifugate at 800 g for 5 min at 4C, respectively. We obtain two 400l solutions from two halves.At this time, take Input, bring the volume to 400l and treat same as with the immunoprecipiated samples. 12- DNA preparation. To 400L extract, add 20g RNase A and incubate for 1 hour at 37C. Then add 16L 5M NaCl, 80g Proteinase K, respectively. Incubate at 65C overnight. 13- The next day, extract with 1 volume of phenol/chloroform/isoamylalcohol and 12000rpm for 15min at room temperature. Take out the supernatant, add 1/10V 3M CH3COONa (pH 5.2) and adequately mix; add 2L DNAmate or glycogen and adequately mix; add 2.5V -20C cold EtOH and mix vigorously and place at -80C for at least 2 hour (or overnight). 12000rpm, 20min, 4C. Washing pellets with -20C cold 70% EtOH; 12000rpm, 15min, 4C. Tip out the supernatant carefully, air dry and resuspend in 50 L TE. 14- Analysis From the 50 l of purified DNA, take 1l of inputs or samples respectively and perform PCR. ChIP-3C ProtocolVolumes are given for 5 x 106 K562 Cells.1- Collect cells and tip out one extra volume cells for estimation of cell number. Then directly fix the cells in the culture medium to a final concentration of 1% formaldehyde and incubate for 15 min at Room Temp.2- Stop Cross-link.Add Glycine to final concentration of 0.125 M. Incubate for 5 min. Wash the cells twice with 5 mL cold PBS. Collect the cells in 1 ml cold Cell Collection Buffer. Incubated on ice for 15 min and subsequently at 30C for 15 min.3- Preparation of Cell nuclei/Chromatin fragmentation with SDS derived method sequentially by vortexing.Spin Cells 5 min at 2000 g Washings/Resuspend:Wash/Spin 3000 g 5 min at 4C each time.1 ml of cold PBS1 ml of NCP Buffer I1 ml of NCP Buffer IILysis: Resuspend in 350 ml of Lysis Buffer, incubate on ice30minSonicate 79 times 20 seconds at 200W output, with 1 minutes refractory period between sonications. Spin 10 min at 12000 g to save soluble chromatin.4- Add 1000U restricted enzyme and EcoRI Buffer to soluble chromatin and incubate at 37 C for overnight. Note: EcoRI enzyme should be high concentration one purchased from NEB. 5- Dilute 300l lysate (5*106 cells) to 2ml using IP Buffer.6- Preclearing.Add:4g IgG and 35l 50 % Protein A/G-agrose beads slurry.Incubate 2-3 hours at 4C under rocking.7- Pellet the unspecific complexes by centrifugation at 800g for 5 min at 4C8- Immunoprecipitation:Add 4g IgG or specific antibody.Incubate under rocking overnight at 4C.The next morning add 35 l 50 % Protein A/G-agrose beads slurry.Incubate 4 hours at 4C under rocking.9- Pellet the s