probing global binding of SDG8 to its targets using HA-SDG8 trangenic lines
This experiment is to determine the global binding of SDG8 to its genomic targets. To do this, HA-tagged SDG8 (hSDG8) was transformed into sdg8-5 deletion mutant background. hSDG8 plants were used for the HA-tagged SDG8 binding profiling by ChIP-seq, where an anti-HA antibody (Abcam) was used to pull down the HA-tagged SDG8 in chromatin samples. The plants were grown in 1% agar plates with 1X Basal MS medium (GIBCO Formula# 97-5068EC) supplemented with 1% sucrose, 2 mM KNO3 and 0.5g/L NaMES at a pH of 5.7 for 2.5 weeks under 16h 130 uE m-2s-1 Light/ 8h dark cycles at 22°C. ChIP-seq was performed using shoots samples.  Barcoded adaptors and enrichment primers (BiOO Scientific, TX, USA) were used for preparing ChIP-seq libraries according to the manufacturer’s protocol, to be compatible with Illumina Hi-Seq platform. In addition, sdg8-5 and WT were also grown together with hSDG8, and the global H3K36me3 profiling of hSDG8, sdg8-5 and WT were performed by ChIP-Seq, also using Illumina Hi-Seq compatible barcoded adaptors and enrichment primers (BiOO Scientific, TX, USA), to validate if hSDG8 could complement the sdg8-5 mutant phenotype of H3K36me3 hypomethylation. Pooled barcoded libraries were sequenced on an Illumina HiSeq platform for 100 cycles in paired-end configuration (Cold Spring Harbor Lab, NY).     The description of each run is as follows:SRR1636731=hSDG8 anti-H3K36me3 ChIP-seq; SRR1636732=hSDG8 anti-HA ChIP-seq; SRR1636733=hSDG8 input DNA as background (whole cell extract); SRR1636734=sdg8-5 anti-H3K36me3 ChIP-seq; SRR1636735=sdg8-5 input DNA as background (whole cell extract); SRR1636736=Wild type anti-H3K36me3 ChIP-seq; SRR1636737=wild type input DNA as background (whole cell extract);