Anti h2aub

A subcellular protein fractionation kit for cultured cells (Thermo Fisher Scientific, Waltham, MA, USA; 78840) was used for fractionation experiments according to the manufacturer’s instructions. A 10 cm dish format was applied, which corresponded to a packed cell volume of 20 µL per well. Localization of SAMD1 was determined using a homemade SAMD1 antibody recognizing the SAM domain [9 (link)]. As loading controls for the respective fractions, a homemade SP1 antibody [18 (link)], anti-tubulin (Merck, Kenilworth, NJ, USA; MAB3408), and anti-H2Aub (Cell Signaling Technology, Danvers, MA, USA; 8240) were applied. For the detection of histone marks, the chromatin-bound fraction was used. H3 (Abcam, Cambridge, UK; ab1791), H3K4me2 (Diagenode, Denville, NJ, USA; C15410035), H3K4me3 (Diagenode, Denville, NJ, USA; C15410003) and H3K14ac (Abcam, Cambridge, UK; ab52946) antibodies were applied. The signal was quantified using the ImageLab software (v5.2.1, Bio-Rad, Hercules, CA, USA) and normalized to the H3 signal.

ChIP experiments were performed as previously described [51 (link)]. Chromatin was extracted from 7-day-old whole seedlings (150 seedlings). Anti-H2Aub (Cell Signaling Technology, 8240S) and anti-H3K27me3 (Diagenode, C15410069) antibodies were used for chromatin immunoprecipitation. For ChIP-seq, two immunoprecipitations from independent biological replicates were processed for next-generation sequencing library preparation. All libraries were prepared by the Ovation® Ultralow Library Systems (NuGEN) following the manufacturer’s instruction using 80% of a typical ChIP as starting material. After amplification for 16 PCR cycles, DNA of a size range between 200 and 500 bp was purified from an agarose gel. Amplification was confirmed by testing an aliquot of the library before and after amplification by qPCR. Sequencing was carried out as single-end 100-nucleotide reads on an Illumina HiSeq by the Max Planck Genome Centre in Cologne. For ChIP-qPCR, amplification was performed using Sensi FAST SYBR & Fluorescein kit (Bioline) and an iQ5 Biorad system. Samples were normalized to input DNA prepared from a reverse cross-linked aliquot of each chromatin preparation. qPCR data are shown as the means of two replicates from a representative experiment. Primers used for ChIP-qPCR are shown in Additional file 1: Table S2.

ChIP-reChIP experiments were performed as previously described^{63 (link)}. In brief, 10-day-old WT seedlings were fixed in 1% formaldehyde. Chromatin was extracted from fixed tissue and fragmented using a Bioruptor® Pico (Diagenode) in fragments of 500–1000 bp. ChIP was first performed with anti-H3K27me3 (Millipore, 07-449, dilution 1:300). Immunocomplexes were captures using Protein A Sepharose beads CL-4B (GE Healthcare). After washing the Protein-A beads, one fraction was processed as in a conventional ChIP assay in order to revert the crosslinking and purify the DNA, and another fraction was used to elute the immunoprecipitated protein–DNA complexes in Re-ChIP elution buffer (2 mM EDTA, 500 mM NaCl, 0.1% SDS, 1% NP40) by incubation for 30 min at 37 °C. The eluate was diluted 20 times with ChIP dilution buffer supplemented with 50 μg of BSA and protease inhibitor. Then, the second ChIP was performed with anti-H2Aub (Cell Signaling Technology, 8240S, dilution 1:100). Immunocomplexes were captures using Protein A Sepharose beads CL-4B (GE Healthcare). After washing the Protein-A beads, chromatin was eluted, de-crosslinked, and the DNA was extracted. The DNA obtained in the two sequential ChIPs were used as PCR template to amplify PLT5, SOC1, and AG using specific primers (Supplementary Table 1).