Instrument: Illumina HiSeq 2000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: Formaldehyde cross-linking and extraction of nuclei was performed as described in Carles and Fletcher (2009) with the following modifications: infiltration time was prolongated (6 min and 24 min), filtration of the extract after grinding was performed through 75 µm and 50 µm nylon meshes and sonication was done for 13x 30 sec on, 1 min off, position H (this longer sonication was only performed for biological replicate 2). Immunoprecipitation reactions were set up as described in Carles and Fletcher (2009) except that 15 µg of cauliflower and 10 µg of wild type chromatin was used and the chromatin was incubated with 5 µl of the antibody alone for 5 hours prior to addition of Protein A Agarose beads without salmon sperm DNA (Millipore). Washing of the ChIP reaction was performed using binding/wash buffer as described in Carles and Fletcher (2009) but for four times 10 min at 4°C and once 10 min at room temperature (RT). Elution and reverse cross-linking was performed as described in Kaufmann et al. (2010) except that the elution was done by vortex mixing for 1 min at RT and input samples (1/3 of the chromatin used for one ChIP sample) were brought to the same volume by adding elution buffer and Tris pH9 in the same ratio as to the ChIP samples. Reverse cross-linked samples were purified using the MinElute Reaction Cleanup Kit (Quiagen) and DNA was eluted in 30 µl Elution buffer. For biological replicate 2, three technical replicates of ChIP on the same nuclear extract were performed and pooled on one MinElute column. Libraries from ChIP samples of Replicate 1 were prepared and sequenced on a GAIIx (Illumina) sequencer as described previously (Yant et al., 2010; Immink et al., 2012). For biological replicate 2, ChIP-seq library preparation was performed using the Truseq ChIP sample preparation kit (Illumina, ref. IP-202-1012) according to the manufacturer's instructions except that PCR amplification was performed prior to size selection. Briefly, sonicated chromatin was repaired prior to 3' ends adenylation. Illumina's indexed adapters were ligated to the adenylated double-stranded DNA fragments. A 18-cycles PCR was performed on the ligated DNA using Illumina's PCR primers. The libraries were then size-selected between 300 and 700 bp on a 2% agarose gel. Each library was validated using high sensitivity chip (Agilent, ref. 5067-4626) on an Agilent Bioanalyzer and quantified using the Kapa library quantification kit (Clinisciences, ref. KK4824). Equimolar amounts of 4 libraries were pooled, diluted to 10 nM, denatured, and diluted again to 7 pM. 100 µl of the diluted pool were hybridized on one lane of an Illumina's Flow Cell. Clustering and 50 cycles sequencing were performed according to Illumina's instructions on a Hiseq2000. Libraries were multiplexed by three (H3K4me3 samples) or four and sequenced on a HiSeq2000 (Illumina) sequencer, yielding 50 bp single-end reads.