GSM1528704: ChIP rep3; Oryza sativa; ChIP-Seq
Chromatin from approximately 1.6g of mature leaf tissue or 100 mg of immature panicle stage In7 was used for each immunoprecipitation with H3K27me3 antibody (Millipore 07-449). Chromatin Immunoprecipitation was performed essentially according to (Bowler et al., 2004) with the following exceptions. All three extraction buffers contained 1mM PMSF and lacked protease inhibitors. The nuclei lysis buffer was modified from (Saleh et al., 2008) with only 0.1% SDA and no protease inhibitors. Chromatin was sonicated on the high setting of a Bioruptor UCD-200 (Diagenode) for 8 thirty-second cycles to produce approximately 500 bp fragments. Immunoprecipitation was performed using Pierce Protein A/G Magnetic Beads (Thermo Scientific). Elution and reverse crosslinking of chromatin was performed simultaneously for 2 hours at 65oC. ChIP purified DNA was amplified using the Sigma Genomeplex Whole Genome Amplification (WGA2) kit following the manufacturer’s protocol (Sigma-Aldrich Co.).  Quantification of ChIP enrichment was performed by Quantitative PCR with ChIP and input (no IP) DNA samples. Primers were designed using the Beacon Designer software (BioRad). The rice ACTIN gene (Os03g50885) was utilized as a negative control for H3K27me3 with the following primers: Actin ChIP qPCR Forward: 5’-GCTGTTTGGATCTCAGGGTGGTTTC-3’ Actin ChIP qPCR Reverse: 5’-GCCGACAATGCTGGGGAAGAC-3’. Primers used to assayed OsMADS1, OsMADS22 and OsMADS34 are as follows: OsMADS1 qPCR For: 5’-GTAGCCAAACCACACCACCATAAAG-3’ OsMADS1 qPCR Rev: 5’-ATCTTCTTCCTCCTCCTCTCCTCTC-3’. OsMADS22 qPCR For4: 5’-GCCTTTGCCAGTTGCGGTGTTG-3’ OsMADS22 qPCR Rev4: 5’-GAGGGTGAGGTGAGGTGAGGTGAG-3’. OsMADS34 qPCR For: 5’-GTTTGTGTTGATTTGCTGGTCTGCTAGAG-3’ OsMADS34 qPCR Rev: 5’-CGATCCGCTGAAGCACCACCTTG-3’. OsMADS6 primers (MADS1506f and MADS1645r) can be found in (Zhang et al., 2010). Fold enrichment was calculated relative to ACTIN. ChIP DNA samples described above were prepared for high throughput Illumina sequencing using the NuGEN Ovation Ultra Low DR Multiplex System 1-8 (NuGEN). 1ug of amplified ChIP DNA for each sample was sheared using the Covaris S220 Focused Ultrasonicator (Covaris Inc.) and approximately 166ng of this sheared DNA was put into each library. One hundred cycles of single read sequencing was performed on the Illumina HiSeq2000 machine (Illumina).
GEO Accession
GSM1528704