Plant Materials and Growth Conditions
Harvest young fresh leaves (~2 grams) prior to bolting (6-8 rosette leaves in A. thaliana, 10-12 leaves in A. arenosa, and 12-15 leaves in allotetraploids). Grow plants under 16/8 hours (light/dark) cycles and harvest samples at Zeitgeber time 6 (noon), unless noted otherwise.
ChIP Antibodies
• Anti-CCA1 antibody (Elaine Tobin, University of California, Los Angels, also made by the Chen Laboratory at The University of Texas at Austin)
• Anti-dimethyl-H3-Lys4 antibody (Upstate, 07-030)
• Anti-trimethyl-H3-Lys4 antibody (Abcam, ab8580)
• Anti-dimethyl-H3-Lys9 antibody (Upstate, 07-521; Abcam, ab1220)
• Anti-dimethyl-H3-Lys27 antibody (Abcam, ab24684)
• Anti-trimethyl-H3-Lys27 antibody (Abcam, ab6002)
• Anti-acetyl-H3-Lys9 antibody (Upstate, 07-352)
Stock Solutions and Buffers
100mM PMSF: Dissolve 0.0871 g PMSF in 5 ml ethanol (note: PMSF is unstable in aqueous solution, always add it fresh to extraction buffer prior use).
Extraction buffer 1: 0.4 M sucrose, 10 mM Tris-HCl pH8, 10 mM MgCl2, 5 mM β-mercaptoethanol, 1 mM PMSF, protease inhibitor cocktail tablet (1 tablet/10 ml solution) (note: freshly add β-mercaptoethanol, PMSF and protease inhibitor cocktail tablet before use).
Extraction buffer 2: 0.25 M sucrose, 10 mM Tris-HCl pH8, 10 mM MgCl2, 1% Triton X-100, 5 mM β-mercaptoethanol, 1 mM PMSF, protease inhibitor cocktail tablet (1 tablet/ 10 ml solution).
Extraction buffer 3: 1.7 M sucrose, 10 mM Tris-HCl pH8, 2 mM MgCl2, 0.15% Triton X-100, 5 mM β-mercaptoethanol, 1 mM PMSF, protease inhibitor cocktail tablet (1 tablet/10 ml solution).
Lysis buffer: 50 mM Tris-HCl pH8, 10 mM EDTA, 1% SDS, 1 mM PMSF, protease inhibitor cocktail tablet (1 tablet/10 ml solution) (note: include 10mM sodium butyrate if evaluating histone acetylation; sodium butyrate inhibits histone deacetylation).
ChIP dilution buffer: 16.7 mM Tris-HCl pH8, 167 mM NaCl, 1.1% Triton X-100, 1.2 mM EDTA, protease inhibitor cocktail tablet (1 tablet/10 ml solution) (note: include 10 mM sodium butyrate if evaluating histone acetylation).
Low salt buffer: 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH8.
High salt buffer: 500 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH8.
LiCl wash buffer: 0.25 M LiCl, 1% Nonidet-P40 (NP40), 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH8.
Elution buffer: 0.1 M NaHCO3, 1% SDS (Note: prepare the buffer fresh and heat up to 65°C prior to use).
TE buffer: 10 mM Tris-HCl pH8, 1 mM EDTA