Bisulphite Conversion, Sequencing, and Evaluation After treatment with RNase A and proteinase K, 1–2 mg of genomic DNA were digested overnight with BamHI (MBI Fermentas). Subsequent bisulphite conversion was carried out using the Epitect Conversion Kit (Qiagen) and controlled for completion as described [21,53]. Converted DNA was used for PCR amplification. PCR-amplified DNA was cloned using pGEM-Teasy (Promega) and ligation mixes transformed into DH5a cells (Invitrogen) and sequenced by terminal-labeling using BigDye Terminator v3.1 (Applied Biosystems). The sequence information obtained was analyzed with CyMATE, www.gmi. oeaw.ac.at/cymate [54], and Excel (Microsoft). Chromatin Immunoprecipitation ChIP was performed as described (http://mescaline.igh.cnrs. fr/EpiGeneSys/www/images/protopdf/p13.pdf) using 3 week- old seedlings. The chromatin was immuno-precipitated with antibodies to histone H3 (Abcam, ab1791), H3K4me3 (Upstate, 07-473), H3K9me2 (T. Jenuwein 4677 and Abcam ab1220), and H3K27me2 (Upstate, 07-473). Immunoprecipitated DNA was purified using a Qiagen PCR Purification Kit and eluted in 50 ml of EB buffer. Quantitative real-time PCR was carried out in a total reaction volume of 25 ml and qPCR conditions were according to the 26 SensiMix Plus SYBR & Fluorescein Kit (Quantace) protocol using an iQ5 Real-Time-PCR System (BioRad Laboratories). qPCR data were evaluated as a ratio to input DNA [55]. sRNA Isolation, Library Generation, and Bioinformatic Analysis Small RNA was isolated from either pooled inflorescences or seedlings (21 days old) using the mirVana miRNA Isolation Kit (Ambion). Small RNA libraries were generated as previously described [56] and sequenced using the Illumina G2 platform. After clipping the adapter sequence by vectorstrip software from EMBOSS package [57], small RNA reads were screened for homology with the epiallele sequence using bowtie [58], allowing only perfect matches (Table S3). Reads homologous to tRNA, rRNA, snRNA, snoRNA, mitochondrial RNAs, and chloroplast RNAs were removed by custom Perl scripts. The total number of reads that mapped to a certain region was computed as sum of 1/ N_i (N_i is the number of times the read i was mapped). It was then normalized to indicate the number of each read per million bp (adapted from the RPKM concept in RNA-Seq, [59]. A threshold of 10 reads was chosen for any sequence to be taken into account. For the epiallele region, the normalized number of mapped reads was computed at single bp scale. For a more detailed view on a selected region, the analysis was performed with SiLoMa [60]. Additional methods are described in Text S1. Supporting Information Figure S1 Schematic representation of the origin of the epialleles. Protoplast culture of transgenic, diploid and hygro- mycin-resistant line C [25] and regeneration resulted in tetraploid plants without (red) or with (blue) hygromycin resistance. The tetraploids were diploidized by repeated backcrossing to diploid wild type and subsequent selfing to generate homozygotes. (TIF) Figure S2 Histone modifications within epialleles and flanking regions. Histone H3 modifications were analysed at eight positions by chromatin immunoprecipitation using antibodies against Genetic Rearrangements Modify Nearby Chromatin H3K4me3, H3K9me2 and H3K27me2. S, inactive epiallele; R, active epiallele; W, wild type. (TIF) Figure S3 Localization of epialleles in interphase nuclei. To investigate whether the epigenetic state had any influence on the location of the epialleles within the nucleus we performed fluorescence in situ hybridization (FISH) on flow-sorted interphase nuclei from S and R lines and wild type. We used different BAC probes on chromosomes 1 and 3 (A). The genomic location of the epiallele and two control regions with equal distance to the telomeres were marked by two differently labelled neighboring BAC clones each. We determined the percentage of nuclei with one (B) or two (C) signals, indicating pairing or non-pairing of corresponding regions (D). We further examined the intranuclear localization (E) and the co-localization with centromeric hetero- chromatin (180 bp repeats, E) of the epialleles. No significant differences between S, R, and wild type were observed, indicating that the expression state did not modify the position within the nucleus or the association with heterochromatin. Bar = 5 mm. S, inactive epiallele; R, active epiallele; W, wild type. (TIF) Figure S4 Maintenance of epigenetic modifications at epialleles in callus culture. (A) DNA methylation analysis at promoters P1 and P2 in callus tissue by bisulfite sequencing representing total (mC) and sequence context-specific (mCG, mCHG, mCHH) methylation in plant tissue and dedifferentiated callus. (B) Methylation analysis of callus tissue DNA treated with HpaII not cutting mCmCGG, blotted and hybridized to a probe spanning the P1 transcript. (C) Histone H3 modifications at promoter duplications analysed in callus tissue by chromatin immunopre- cipitation using antibodies against H3K4me3, H3K9me2 and H3K27me2. S, inactive epiallele; R, active epiallele; W, wild type. (TIF) Figure S5 Analysis of antisense transcripts. The overlap between the short and the long transcript from the two promoters suggested a search for non-coding RNAs involved in silencing maintenance. To investigate whether the non-coding sequence (NC) down- stream of P2 could have served as a promoter to produce antisense RNA from the epiallele, we cloned the NC fragment in both orientations in front of a GUS reporter gene, replacing the P35S promoter in vector pCBK04. We then tested the constructs by transient transformation via Agrobacterium tumefaciens of an Arabi- dopsis Col-0 cell suspension culture and screened for GUS expression (A). None of the constructs gave any indication of GUS expression, making a promoter-like function of the NC sequence unlikely. Further, we analyzed potential antisense transcripts by northern blot hybridization with labeled strand- specific oligonucleotides homologous to different regions (P1/P2, HPT, V1/V2, NC) of the epiallele (B). Control sense and anti- sense RNA included in the blots were generated by in vitro T7 or SP6 polymerase transcription of the respective sequences cloned in the pGEM-T easy vector (Promega). No specific antisense RNA from the epiallele could be detected. This negative result was further confirmed for S and R lines by RT-PCR with primers at three different positions (data not shown). S, inactive epiallele; R, active epiallele; W, wild type. (TIF) Figure S6 Analysis of polyadenylation. Analysis of polyadenyl- ation by northern blot hybridization of total (A,C,E) and poly(A)- enriched (B,D,F) RNA from cis-mutants in comparison to S (inactive epiallele) and R (active epiallele). * RNA sample degraded. (A,B) Probe specific for P1 transcript (HPT, PLoS Genetics | www.plosgenetics.org 11 October 2011 | Volume 7 | Issue 10 | e1002331