TITLE: Effective identification of DNA-bound protein complexes using Chromatin Immunoprecipitation
AUTHORS: Georgios I Laliotis, Philip N. Tsichlis

[STEPS]
?. [Chemicals Required]
1. Cytosolic Lysis Buffer (200 ml) ; 5 mM PIPES (pH 8.0),  85 mM KCl, 0.5% NP40 + PI     PIPES 0.3 g   =>Adjust pH 8.0          2M KCl          8.5 ml         10% NP40      10 ml 2. Nuclear Lysis Buffer (50 ml) ; 50 mM Tris (pH 8.0),  10 mM EDTA, 0.5% SDS          1 M Tris (pH 8.0)             2.5 ml          0.5 M EDTA                      1 ml          20% SDS                       1.25 ml 3. IP Dilution Buffer (250 ml) ; 16.7 mM Tris (pH 8.0), 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton X-100, 0.01% SDS       1 M Tris (pH 8.0)             4 ml           0.5 M EDTA                 0.6 ml           5 M NaCl                      8.5 ml           20% Triton X-100       12.5 ml           10% SDS                     250 ul  4. Low Salt Wash Buffer (250 ml); 20 mM Tris(pH8.0), 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.1% SDS     1 M Tris (pH 8.0)           5 ml            0.5 M EDTA                   1 ml            5 M NaCl                     7.5 ml            20% Triton X-100      12.5 ml           10% SDS                     2.5 ml 5. High Salt Wash Buffer (250 ml); 20 mM Tris(pH8.0), 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% SDS      1 M Tris (pH 8.0)            5 ml             0.5 M EDTA                   1 ml             5 M NaCl                      25 ml             20% Triton X-100      12.5 ml             10% SDS                     2.5 ml 6. NS Buffer ; 50 mM Hepes, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS 7. LiCl Wash Buffer (250 ml); 10 mM Tris (pH8.0), 1 mM EDTA, 250 mM LiCl, 1% NP40, 1%(w/v) deoxycholic acid     1 M Tris (pH 8.0)           5 ml        0.5 M EDTA               0.5 ml            1 M LiCl                    72.5 ml             10% NP40                   50 ml            Deoxycholic acid        0.25 g 8. Miscallenous     1.25 M Glycine              9.38 g / 100 ml     1 M NaHCO3                 8.4 g / 100 ml       =>   aliqute (0.5 ml) and keep at -20 °C
?. [Cross-linking and Lysis of the cells]
For each Ab to be tested, 3 plates for each cell line must be used.Add 270 ul of 37% Formaldehyde into 10 ml culture media (P100)Incubate at 37 °C for 15 minAdd 1 ml of 1.25 M Glycine and incubate 5 min at RTWash with cold PBS (x2)Wash with 1 ml of cold PBS (+ PI)Scrap cells and collect into a new tubeTip : Scrap each plate and place it in a different tubeCentrifuge at 14000 rpm for 1 min at 4 °C to remove PBSRemove supernatantIncubate lysis buffer in 37 deg. For a few minutes to dissolve precipitantsAdd fresh 1x proteinase inhibitor-10ul from 100x stockAdd/Resuspend 1 ml of nucleic lysis buffer (+PI) per 3 P100 plates ==> 200ul for  1x106Manually shake tube to break pelletSplit the 1ml lysate in 2 tubes with 500ul-Helps the sonication processIncubate on ice for 10’Sonication : 15 sec on/45 sec off  (60 sec x 6-7 times = 6-7 min on/each sample)                 at 30% duty 17. Centrifuge at 14000 rpm for 15 min at 4 °C18. Transfer supernatant into a new single tube         * can be stored at -80 °C         * keep 10% lysate (50ul-10% of 500ul Ab solution) for input-you can dilute it to 1% I put for qPCR analysis
?. [Immunoprecipitation of Crossliked protein/DNA complex]
1. Dilute 5 fold of lysate volume with IP dilution buffer in a 15 ml tube2. Add 1x protease inhibitors in the IP dilution buffers 3. Dilute to 5ml from initial 1ml using 4ml IP dilution buffers4. Split in 2x tubes for Ab and 2 for normal IgG           => final buffer concentration) 0.1% SDS of nucleic lysis buffer 5. Pre-cleaning : Add 30 ul of protein G bead / Ab reaction for pre-clearing per tube6. Incubate for 1 hr at 4 °C with rotation7. Centrifuge at 3000 rpm for 2 min at 4 °C8. Transfer supernatant into a new 2 ml tube 9. Add 2 ug of antibody or normal IgG per tube (for Normal Rabbit IgG add 2ul from 1mg/dL. In order to make that add 8.77ul from 11.4mg/dl stock and 92.23 ddH20)10. Incubate overnight at 4 °C with rotation11. Add 25 ul of protein G agarose bead / Ab reaction per tube12. Incubate for 2 hrs at 4 °C with rotation13. Centrifuge at 3000 rpm for 2 min at 4 °C14. Place beads at least 5 minutes in magnetic rack15. Remove supernatant carefully with WB long tips gradually16. Add/Resuspend beads 500 ul cold low salt wash buffer/tube and combine tubes in 117. Rotate for 5’ at 4 deg18. Wash with wash buffer            Order : cold low salt wash buffer (x2)             cold high salt wash buffer (x2)              cold LiCl wash buffer (x2)             cold TE buffer (x3)19. Centrifuge at 3000rpm for 2’20. Place on magnetic rack for 5’21. Add 500ul of buffer22. incubate for 5 min at 4 °C with rotation23. Centrifuge at 3000 rpm at RT (or 4 °C) for 1 min24. Place on magnetic rack for 5’25. Remove supernatant26. Wash with next one etc.27. Add 200 ul elution buffer (0.1M NaHCO3and 1% SDS)For 1.5ml : 1,2ml H20 firstthen, 150ul 10x SDSfinally, 150ul 1M NaHCO328. Incubate for 15 min with shaking heating block (900 rpm) at 23 °C29. Centrifuge at 3000 rpm for 3 min30. Put in magnetic rack for 5’-Transfer supernatant into a new tube31. Repeat #16-19 (Total elution vol. will be 400 ul)
?. [Reverse Crosslinking and Elution of Protein/DNA complexes]
32. Add 20 ul of 5M NaCl and 1 ul of 20 mg/ml RNase / 200 ul tube (remember the volume is 400ul)          * Don’t forget input sample (10% lysate + 350ul Elution buffer)!! 33. Mix well manually before putting into the cycler34. Incubate at 65 °C in Thermo cycler for 5 hrs or overnight with 450rpm rotation to prevent precipitation35. Add 10 ul of 0.5M EDTA, 20 ul of 1M Tris (pH 8.0), and 2 ul of 10 mg/ml Proteinase K/ tube36. Incubate at 45 °C for 1 hrQIAGEN PCR extraction kit (Or use P/C/I extraction method)37. Add 5 vol. of PBI buffer (QIAGEN PCR extraction kit) and mix well-450ul ==> 2,25ml Final volume           * Check PH => add 10 ul 3M sodium acetate if #23 turn orange or violet color.38.  Add sample into a column-max 800ul39. Centrifuge at 14000 rpm for 1 min 40. Add 750 ul of PE buffer41. Centrifuge at 14000 rpm for 1 min and remove liquid from a tube42. Centrifuge at 14000 rpm for 1 min again43. Add 60 ul of EB buffer44.Centrifuge at 14000 rpm for 1 min 45.Store DNA elution at -20 °C46. Carry out qPCR using  2 ul elution/reaction