GSM1669410: rosettes_wildtype_peg_H3T3ph_rep2; Arabidopsis thaliana; ChIP-Seq
Watered or water deficit treated vegetative tissues were fixed in 1% formaldehyde for 10 min after vacuum infiltration of the tissues in phosphate buffered saline (PBS) with 0.007% silwet. Fixation was stopped by quenching with 125 mM glycine. Next, the tissue was rinsed twice in ice cold water, blotted dry and flash frozen in liquid nitrogen. Chromatin was isolated and resuspended in 300 ml nuclei lysis buffer and sonicated for four 15 sec pulses using a Branson sonifier 450 at an output between 10 – 15%. 100 ml of sheared chromatin was diluted in 900 ml ChIP dilution buffer and precleared, using 5 ml of protein A Dynabeads (Invitrogen, Carlsbad, CA). Protein A beads were removed on a magnet, and the precleared chromatin was incubated overnight at 4 °C with antibodies against either mono-methylated H3K4 (10 mL, Abcam ab8895), dimethylated H3K4 (6 mL, Upstate 07-212), or trimethylated H3K4 (6 mL, Abcam ab8580). Next the antibody/chromatin complex was precipitated by incubating the mix with 40 ml protein A beads for 1 hour followed by collection of the beads on a magnet. The beads were washed and chromatin eluted and de-crosslinked using procedures. DNA was recovered by phenol/chloroform extraction and ethanol precipitation in the presence of Novagen pellet paint (CN Bioscience) and resuspended in 30 mL of TE buffer. Purified DNA was end modified, ligated to amplification primers, amplified, and sequenced according to the manufacturer’s protocols (Illumina, San Diego, CA).
GEO Accession
GSM1669410