GSM1247501: JAG-GFP replicate 3; Arabidopsis thaliana; ChIP-Seq
pJAG:JAG-GFP jag-2 and wild-type L-er control plants were used. 1300-1500 mg  (fresh weight) of inflorescence apices per sample were fixed in 35 ml fixation buffer (0.4 M sucrose, 10mM Tris pH 8, 1 mM EDTA pH 8.5, 1% formaldehyde, 100 µM PMSF) under vacuum for 20 min on ice. Cross-linking was stopped with 100 µM  glycine for 10 min on ice. After two washes with sterile water, the tissue was blotted dry and frozen in liquid nitrogen. Nuclei were purified as described [Gomez-Mena et al., 2005, Development 132: 429-438] and resupended in 1 ml of sonication buffer containing 500 mM Hepes, 150 mM NaCl, 5 mM MgCl2, 10% TRITON X-100 and ½ a tablet of protease inhibitor cocktail complete Mini, EDTA-free (Roche). Sonication was performed in a Bioruptor waterbath sonicator at 4°C (2 x 5 min high power level with 30 sec on/30sec off cycles) resulting in an average fragment size of 500 bp.  After centrifugation, the supernatant was mixed with 500 ul of immunoprecipitation buffer containing 0.5M Hepes, 150 mM NaCl, 5 mM MgCl2 , 10% TRITON X-100, 1 mg/ml BSA and 25 µl of anti-GFP µMACS Microbeads (Milteyi Biotec), incubated on ice for 30 min and loaded on a µ Column (Milteyi Biotec) that had been equilibrated with 200 µl immunoprecipitation buffer and placed into a magnetic µMACS separator (Milteyi Biotec). After washing twice with 400 µl and twice with 200 µl of immunoprecipitation buffer, then twice with 200 µl TE buffer (100 mM Tris pH 8, 10 mM EDTA pH 8), DNA was eluted once with 20 µl and twice with 50 µl of preheated (96° C) elution buffer containing 50 mM Tris pH8, 10 mM EDTA, 50 mM DTT and 1% SDS. 100 µl of TE-buffer and 9 µl of 25 mg/ml Proteinase K (Sigma) were added to the eluted samples and to the input control samples. Crosslinking was reverted in eluted and input samples at 37°C overnight, followed by addition of 9 µl of 25 mg/ml Proteinase K and 8 h incubation at 65° C, phenol-chloroform extraction and precipitation with ethanol overnight at -20° C. After washing in 70% EtOH, the air-dried DNA was resuspended in 100 µl PCR-grade water (Roche), purified using a PCR purification Kit (18104, Qiagen) and stored at -80° C. Illumina TruSeq ChIP-Seq libraries (three pJAG: JAG-GFP replicates and three wild-type controls) were produced as described [Kaufmann et al, 2009, PLoS Biology 7: e90.]
GEO Accession
GSM1247501