title: GSM5179614: fve_H3Ac_rep2 [ChIP-seq]; Arabidopsis thaliana; ChIP-Seq
library_construction_protocol: Two gram of 11-day-old Arabidopsis seedlings was ground in liquid nitrogen to powder and then homogenized in lysis buffer. After 5 min of rotation at 4°C, 208 μl of formaldehyde was added to fix the materials for 20 min and then 469 μl of 2 M glycine was added to terminate the reaction. The homogenate were filtered through 4 layers of Miracloth (Millipore) and pelleted by centrifugation at 1,500 g for 10 min at 4°C. The pellet was washed 4 times with NRBT buffer and resuspended in 400 μl of lysis buffer. The pellet was sonicated (30 s on /30 s off cycle) for 8 min and 3 times. After sonication, the chromatin was centrifuged at 4°C for 15 min at 13,000 g. The supernatant was then dilute 5 times with dilution buffer. The chromatin was incubated with 40 μl of Protein A for 2 h at 4°C to remove non-specific binding and then immunoprecipitated overnight with antibody. An additional 40 μl of Protein A was added to conjugate the antibody for 2 h. Thereafter, the resin was washed with low salt wash buffer, high salt wash buffer, LiCl wash buffer, and TE wash buffer. The DNA-protein complex was eluted twice from the resin with 200 μl of elution buffer. 24 ul of 5 M NaCl was added to 400 μl of the elution to reverse cross-linking for more than 10 h. After digesting the protein with proteinase K, the DNA was purified and then subjected to library preparation and Illumina sequencing. Libraries were built for ChIP-seq (Illumina HiSeq X Ten; 150-bp pair-end sequencing).
attributes.tag: GEO Accession
attributes.value: GSM5179614