GSM1260848: LHY_ChIPSeq; Arabidopsis thaliana; ChIP-Seq
Samples were cross-linked by vacuum-infiltration in 1% formaldehyde for 10 min. The cross-linking reaction was quenched by adding 1.3 mls of 2 M glycine and infiltrating for a further 5 min. Each sample was homogenised in 30 mls of Extraction Buffer 1 (0.4 M sucrose, 10 mM Tris-HCl pH 8, 5mM beta-mercaptoethanol, 1 mM PMSF and protease inhibitors (Roche complete protease inhibitor cocktail)) using a Polytron Status X120. Debris was removed by filtration through miracloth and the sample was centrifuged at 4°C. The resulting pellet was gently resuspended in 1ml of Extraction Buffer 2 (0.25 M sucrose, 10 mM Tris-HCl pH 8, 10 mM MgCl2, 1% Triton X-100, 5 mM beta-mercaptoethanol, 1 mM PMSF and protease inhibitors) and the nuclei collected by centrifugation. The nuclei were lysed in 500 µl of Nuclear Lysis Buffer (50 mM Tris-Hcl pH 8, 10 mM EDTA, 1% SDS, 1 mM PMSF and protease inhibitors) and sonicated to give chromatin fragment sizes ranging from 100 to 1000bp. For each immunoprecipitation, 250 µl of chromatin was added to 2 mls of ChIP dilution buffer (167 mM NaCl, 16.7 mM Tris-HCl pH8, 1.2 mM EDTA, Triton X-100, 1 mM PMSF and protease inhibitors) and pre-cleared with protein A Dynabeads (Invitrogen). Samples were incubated overnight at 4°C with either anti-LHY (1:200)  antibody. The immuno-complexes were isolated by incubation with protein A Dynabeads for 2 h at 4°C. The beads were washed as described by Haring et al.,  2007 with the addition of three extra high salt buffer washes. Protein A beads were then resuspended in 100 μl of TE and treated with RNase at 37°C for 20 minutes. SDS was added to a final concentration of 0.5% and the samples digested with proteinase K for 2 h at 50°C. To reverse cross-linking, 8 μl of 5 M NaCl was added and the samples were incubated overnight at 65°C. The DNA was then purified using the MinElute PCR purification kit (Qiagen). 9 independent ChIP samples were pooled for the generation of each library.  Wild-type and lhy-21 mutant libraries were sequenced in multiplex. Library preparation and sequencing was conducted at the University of Utah’s Bioinformatic Core facility using an Illumina HiSeq 2000 sequencer. The libraries were prepared using the Illumina Tru Seq DNA sample prep kit according to the instructions of the manufacturer.  Indexes 6 (GCCAAT) and  12 (CTTGTA) were used for wild-type (Ws) and lhy21 samples, respectively.
GEO Accession
GSM1260848