Illumina HiSeq 2500 sequencing; Identification of Peopod2 (PPD2) target genes through GSyellow-based ChIP-seq in Arabidopsis thaliana PSB-D cell cultures.
Identification of Peopod2 (PPD2) target genes through GSyellow-based ChIP-seq in Arabidopsis thaliana PSB-D cell cultures.
Cross-linking of Arabidopsis cell culture material was carried out as described (doi: 10.1104/pp.113.229617 Verkest et al., 2014). Cell suspension cultures were sub-cultured every 7 days by transfering 7 mL culture in 43 mL MSMO medium (containing per liter 4.43 g MSMO (minimal organics Sigma #M6899), 30g Sucrose, H2O, 50 μl kinetin (1 mg/ml stock solution) and 500 μl NAA (1mg/ml stock solution) and a pH adjusted to 5.7 with 1M KOH. Flasks were grown in the dark in an incubator at 25°C shaking at 130 rpm.  ChIP-seq on PPD2-GSyellow was performed using 5 uL ab290 anti-GFP antibody (Abcam), with minor adaptations of a published protocol (Morohashi et al., 2012) and 1 g fixed material was used per ChIP. All subsequent steps were carried out as described by Morohashi et al. 2012. The PPD2-HBH TChAP-seq was performed using an Arabidopsis cell suspension culture overexpressing a HBH tagged PPD2 as described (doi:  10.1105/tpc.15.00006 Gonzalez et al., 2015) ChIP-seq libraries were prepared by GATC Biotech
Experimental Factor: immunoprecipitate
anti-GFP
Experimental Factor: genotype
p35S:PPD2-GSyellow