GSM2302417: GFP_H3K27me3_1; Arabidopsis thaliana; ChIP-Seq
Root protoplast were prepared as described in (Brady et al., 2007). The MoFlo cell sorter’s electronic configuration was modified to identify intact protoplasts above electronic and sample buffer “noise” levels by choosing a side scatter electronic threshold and by applying logarithmic scaling to the forward angle and side angle 488nm laser light scatter signals.  To collect the GFP positive protoplasts, the green fluorescence of the GFP (530/50 detection filter) was separated from the red fluorescence (emission 670/30) of chlorophyl. Protoplast chromatin was crosslinked with 0.1% formaldehyde for 5 min and the reaction was stopped by adding glycine (0.125M final concentration). The chromatin immunoprecipitation assay performed in this study is a modification of the protocol described in (Bouyer et al., 2011). We used four independent biological replicates (100,000 GFP positive protoplast each) and two antibodies: H3K27me3 (Millipore 07-449) and H3K4me3 (Millipore 07-473). DNA recovered after ChIP and the input chromatin were both amplified using SeqPlex Enhanced DNA amplification kit (SEQXE – Sigma) following the manufacturer’s instructions. Amplified DNA was used to synthesize a barcoded Illumina-compatible library (Kumar et al., 2012). Libraries were pooled and sequenced on the HiSeq2000 50SR