ChIPfewtissue.docVIP

CHIP PROTOCOL From Toshiro Ito and Colot, V. (Gendrel, A.V., Lippman, Z., Martienssen, R., and Colot, V. (2005). Profiling histone modification patterns in plants using genomic tiling microarrays. Nature methods 2, 213-218.) Modified by Binglian (Note: at any time, to avoid producing foam when transferring, pipetting) Preparation: Premake protein inhibitor cocktail solution (100-fold stock, 2 tablets into 1ml H2O), keep it on ice and PMSF (100-fold stock, 100 mM) at room temperature Premake Glycine solution (0.75g/5mL Distilled Water) Prepare at least 8 ml/each sample M1 solution with mercapto-ethanol 0.7 ul/ml, protein inhibitor, and PMSF Prepare at least 12 ml/each sample M2 solution with mercapto-ethanol 0.7 ul/ml, protein inhibitor, and PMSF Prepare at least 2 ml/each sample M3 solution with mercapto-ethanol 0.7 ul/ml, protein inhibitor, and PMSF Prepare at least 0.5 ml/each sample SDS lysis solution with protein inhibitor and PMSF Prepare at least 5 ml/each sample ChIP dilution solution with protein inhibitor and PMSF Check protein A agarose/salmon sperm DNA (upstate, Cat.#16-157) Check antibody Conc. For GFP Ab (Cat. #632460, Clontech) 1:200 dilution For H3K9 (#ab1220, abcam) For H3K4 (#ab8580, abcam) For H3K27 (#07-322, upstate) 10. Check these regular chemicals: EDTA, Tris-HCL, NP-40, LiCl, sodium deoxycholate (#D5670, Sigma), Triton X-100, SDS, NaCl, NaHCO3, mercapto-ethanlo, phosphate buffer, hexylene glycol (#M9671, Sigma) DAY 1 ~ 4-5 hrs Carefully grind the inflorescences to fine powder (2 ml for two IP) in liquid N2 and pour the powder into 15 ml yellow tube. Set the tube on ice, add 8 ml M1 buffer and invert several times to mix. Add 216uL formaldehyde (final conc 1%, had better fresh, it’s easily oxidized) and incubate on shaker in cold room for 10 min. Add 543uL 2M Glycine (final conc: 0.125M) to stop the crosslink an