マウス赤血球を用いた ChIP assay（谷本 啓司）
Solutions (050417改訂版)
[Nuclei Swelling Buffer]
5mM PIPES (pH8.0) 100mM PIPES (pH8.0) 2 ml
85mM KCl 1M KCl 3.4 ml
0.5% NP-40 10% NP-40 2 ml
H2O to 40 ml
※Add protease inhibitors before use
[SDS Lysis Buffer]
1% SDS 10% SDS 4 ml
10mM EDTA 0.5M EDTA 0.8 ml
50mM Tris-Cl (pH8.0) 1M Tris-Cl (pH8.0) 2 ml
H2O to 40 ml
※Add PI before use
[ ChIP dilution buffer ]
0.01% SDS 10% SDS 40 μl
1.1% Triton X-100 10% Triton X-100 4.4 ml
1.2mM EDTA 0.5M EDTA 96 μl
16.7mM Tris-Cl (pH8.0) 1M Tris-Cl (pH8.0) 0.668 ml
167mM NaCl 5M NaCl 1.336 ml
H2O to 40 ml
※Add PI before use.
[ Paro Buffer I (low salt) ]
0.1% SDS 10% SDS 0.4 ml
1% Triton X-100 10% Triton X-100 4 ml
2mM EDTA 0.5M EDTA 160 μl
20mM Tris-Cl (pH8.0) 1M Tris-Cl (pH8.0) 0.8 ml
150mM NaCl 5M NaCl 1.2 ml
H2O to 40 ml
[ Paro Buffer II (high salt) ]
0.1% SDS 10% SDS 0.4 ml
1% Triton X-100 10% Triton X-100 4 ml
2mM EDTA 0.5M EDTA 160 μl
20mM Tris-Cl (pH8.0) 1M Tris-Cl (pH8.0) 0.8 ml
500mM NaCl 5M NaCl 4 ml
H2O to 40 ml
[ Paro Buffer III (LiCl, freshly prepared) ]
0.25M LiCl 1M LiCl 10 ml
1% NP-40 10% NP-40 4 ml
1% Sodium Deoxycholate 10% Sodium Deoxycholate 4 ml
1mM EDTA 0.5M EDTA 80 μl
10mM Tris-Cl (pH8.0) 1M Tris-Cl (pH8.0) 0.4 ml
H2O to 40 ml
[ Elution Buffer (freshly prepared) ]
1% SDS 10% SDS 1ml
0.1M NaHCO3 NaHCO3 84mg
H2O to 10ml
[ Protein G Sepharose Fast Flow (Amersham) solution ]
500 μl of Protein G Sepharose beads / Ethanol
Cfg. 12000 rpm, 1’, 4°C (SW)
Removed the sup. (~100μl)
Re-suspended in 800 μl of Dilution Buffer
Cfg. 12000 rpm, 1’, 4°C (SW)
Removed the sup. (~800μl)
Re-suspended in 800 μl of Dilution Buffer
Cfg. 12000 rpm, 1’, 4°C (SW)
Removed the sup. (~800μl)
Re-suspended in 500 μl of Dilution Buffer (total 1ml)
Added sonicated salmon sperm DNA to 1 mg/ml and BSA to 1mg/ml.
100 μl of 10 mg/ml ssDNA
100 μl of 10 mg/ml BSA (Wako; 012-15113, Albumin/Globulin Free-HG)
rotated at 4°C, O/N.
Cfg. 12000 rpm, 1’, 4°C (SW)
Removed the sup. (~800 μl)
Re-suspended in 800 μl of TE.
Cfg. 12000 rpm, 1’, 4°C (SW)
Removed the sup. (~800 μl)
Re-suspended in 800 μl of TE.
Cfg. 12000 rpm, 1’, 4°C (SW)
Removed the sup. (~800 μl)
Re-suspend to original volume (500 μl) of beads in TE buffer.
Add NaN3 to 0.02%. (2 μl of 10% NaN3)
Stored at 4°C.
0. 使用するPBS(-)は4°Cに冷やしておく。以下の操作はことわりがなければ氷上で行う。
1. PH injected Spleen (2~3x108 cells/脾臓半分)
2. 35mmシャーレの中にセル・ストレイナー (FALCON; 35-2350) を入れ、その中にspleen (1/2)を置き、1mlのPBS (-)を加えて、ラバー・ポリスマンでつぶす。
3. シャーレ内に通過した細胞を1mlチップで集め、15-mlコニカル・チューブへ。
4. さらに、2mlのPBS(-)により、細胞を回収し、15-mlチューブへ。(total 3ml)
5. PBS(-)により、total 5mlにfill-upする。(= Single cell suspension)
6. Cell counting (x100 diluted, equal vol. of TB)
[Result (ex.)] 20(cell count)x2(TB)x100(dil.)x104x5ml = 2.0x108 cells/5ml
7. 1.5ml (=6x107 cells) / 36 ml of PBS(-) / 50-mlコニカル・チューブ (6 sample分。つまり、1x107 cellsで1 sample分）を、固定条件の数だけ準備する。
8. 37％(W/W) formaldehydeを1ml加える。（final=1%）
(F79-500/Fisher or 064-00406/WAKO or F1635/Merck/EM SIGMA)
9. Gently shaked in various conditions.（standard: 4°C, 10 min）
10. 2M Glycine solutionを2.3ml加える。
0.375g of glycin (MW=150)/ 2.5 ml of PBS(-) = 2M solution
11. Gently shaked for 5 min at 4°C.
12. Cfg. 3000rpm at RT for 5 min. (Swing-type rotor; SW)
13. Removed the sup.
14. Resuspended in 10 ml of ice-cold PBS(-).
15. Cfg. 3000rpm at 4°C for 5 min. (SW)
16. Removed the sup. by decantation.
17. Repeat 14-16, two more times.
18. Resuspended in 1 ml of Swelling Buffer (-NP-40, +PI) by pipetting.
(PI: proteinase inhibitor ; Roche 1697498; 50 ml with one tablet; freshly prep.ed.)
19. Trnsferred to micro-cfg. (2-ml) tube.
20. Added 50 ml of 10% NP-40 to 1ml of cell suspension. (final conc. = 0.5%)
21. On ice for 10 min.
22. Cfg.ed at 5000 rpm for 5 min at 4°C.
23. Removed the sup.
24. Resuspended in 200μl of Lysis Buffer（広口 tip）.
25. Filled up to 1.5 ml（少し多めに）with Lysis Buffer.
26. Added 30 μl of 25xPI (final 0.5x).
27. 250 mlずつSonication用チューブに分注。(1x107 cells/tube) *泡を入れない。
28. On ice for 10 min.
29. Sonicated the cell suspension. (Bioruptor)
1: 1’ on → 1’ off x3
2: x4
3: x5
4: x6
5: x7
6: x8
30. Cfg.ed at 13200 rpm for 5min at 4°C.
31. Took 180 ml and stored at 4°C.
32. Took 30 ml and incubated for 2 hr at 65C, phenol/CHCl3 extracted (30 ml) and 10 ml of sup. was run on 2% agarose for checking the DNA size.
33. 180 μl of sup. to 2-ml tube (表面さらさら)
34. 1620 μl of ChIP dilution Buffer + 36 μl of 25xP.I.
35. 30-50 μl of Pre-blocked Protein G sepharose beads.
36. Rotated at 4°C, 2h ~ O/N.
37. Cfg.ed at 7000 rpm for 3 min at 4°C. (SW)
38. made 300-500μl aliquots (3~6 tubes)
39. added antibodies
1. No Ab.
2. IgG (~2μg)
3. Ab. 1
4. Ab. 2
5. Ab. 3
6. Ab. 4
40. Rotated at 4°C, O/N.
41. 20μl of Protein G sepharose beads
42. Rotated at 4°C, 2h~.
43. Cfg. 12000rpm at 4°C for 1min. (SW)
44. Removed the sup.
45. Re-suspended in 1ml of Low-Salt Buffer. (0.1xP.I.)
46. Rotated at R.T. for 10’.
47. Cfg. 12000rpm at 4°C for 1min. (SW)
48. Removed the sup.
49. Re-suspended in 1ml of High-Salt Buffer. (0.1xP.I.)
50. Rotated at R.T. for 10’.
51. Cfg. 12000rpm at 4°C for 1min. (SW)
52. Removed the sup.
53. Re-suspended in 1ml of Li-Cl Buffer. (0.1xP.I.)
54. Rotated at R.T. for 10’.
55. Cfg. 12000rpm at 4°C for 1min. (SW)
56. Removed the sup. completely with loading tip.
57. Re-suspended in 1ml of TE.
58. Rotated at R.T. for 10’.
59. Cfg. 12000rpm at 4°C for 1min. (SW)
60. Removed the sup.
61. Re-suspended in 1ml of TE.
62. Rotated at R.T. for 10’.
63. Cfg. 12000rpm at 4°C for 1min. (SW)
64. Removed the sup.
65. Re-suspended in 1ml of TE.
66. Rotated at R.T. for 10’.
67. Cfg. 12000rpm at 4°C for 1min. (SW)
68. Removed the sup. completely with loading tip.
69. 80μl of Elution Buffer.
70. vortexed for 5min at R.T.
71. Cfg. 12000rpm at 4°C for 1min. (SW)
72. took 60μl of sup. to new tube.
73. 60μl of Elution Buffer.
74. vortexed for 5min at R.T.
75. Cfg. 12000rpm at 4°C for 1min. (SW)
76. took 60μl of sup. to new tube. (total 120μl)
77. 2.4μl of 10mg/ml RNase A / 120μl of sample.
78. 55°C for 1h.
79. 6μl of 6mg/ml Proteinase K / 120μl of sample.
80. 65°C, O/N.
このとき、”Input” sampleも65°C.
81. DNA purification with Qiagen PCR purification kit.
1. added 700μl of PB to each sample
2. applied to the column.
3. Cfg.ed at 12000 rpm for 1’.
4. washed with 700μl of PE.
5. Cfg.ed at 12000 rpm for 1’.
6. spin, again.
7. to new micro-cfg. tube.
8. 50μl of Buffer EB.
9. spin, 1min.