GSM1566968: WT Col-0, rep1; Arabidopsis thaliana; ChIP-Seq
The entire experiment from seed sowing through deep sequencing was performed twice to produce independent biological replicates. ChIP-seq was performed with an antibody raised in rabbit (#4028) against the LFY C-terminal amino acids 223-424 (BioGenes, Berlin, Germany). Briefly, 15-day-old 35S:LFY (pCA26) and 35S:LFYTERE (pCA29) and Col-0 (control) seedlings grown under long day photoperiods at 23 °C on MS plates were harvested and fixed as described previously. Frozen tissue was ground, filtered three times through Miracloth (Calibrochem), and washed as described previously thorough buffers M1, M2, and M3. Nuclear pellets were resuspended in sonic buffer as described (1 mM PEFA BLOC SC (Roche Diagnostics) was substituted for PMSF), split into technical duplicate samples, and sonicated with a Branson sonifier at continuous pulse (output level 3) for 8 rounds of 2 x 6 seconds and allowed to cool on ice between rounds. IP reactions were performed by incubating chromatin with 2.5 μl anti-LFY serum overnight at 4 °C as described. The immunoprotein-chromatin complexes were captured by incubating with protein A-agarose beads (Santa Cruz Biotechnology), followed by consecutive washes in IP buffer and then elution as described. Immunoprotein-DNA was then incubated consecutively in RNase A/T1 mix (Fermentas) and Proteinase K (Roche Diagnostics) as described, after which DNA was purified using Minelute columns (Qiagen). ChIP samples were tested for enrichment by QPCR. Deep sequencing libraries were produced by standard Illumina protocols.