Small RNA blot hybridization Total RNA was isolated from 600 mg inflorescence tissue by grinding in liquid nitrogen followed by TRIzol extraction (Invitrogen). 100 mg total RNA sample was size-fractionated on RNeasy Mini Columns (Qiagen) as in Blevins et al. (2006) [62], then 8 mg low molecular weight RNA was separated on an 18% polyacrylamide gel. Probes for specific RNA species were DNA oligonucleotides end-labeled with c[32P]-ATP: the siR1003 probe described in Xie et al. (2004), U6 snRNA probes in Blevins et al. (2006) [62], and miR-160 probe 59-TGG CAT ACA GGG AGC CAG GC A`-39. DNA oligo probe hybridization was performed as in Blevins et al. (2006). The 5S rRNA 39-flank probe was generated by T7 polymerase transcription of a PCR fragment in the presence of a[32P]-CTP. Primers for this template were: T7_5SUNIV2, 59- taa tac gac tca cta tag gga gaC GAA AAG GTA TCA CAT GCC-39 and 39flank_F, 59-GCA TCC CTC TTT TAT GTT TAA CCT- 39. RNA probe hybridization was performed as in Onodera et al. (2005) [7]. DNA methylation assays Genomic DNA was isolated from Arabidopsis inflorescences using Nucleon PhytoPure DNA extraction kit (Amersham). 3 mg aliquots of DNA were digested overnight with 30 U of Alu I, Hpa II or Hae III in the corresponding commercial buffer (New England Biolabs). Following size-separation by 0.8% agarose gel electrophoresis, gel- bound DNA was depurinated, denatured and transferred to Hybond-N+ nylon membrane (Amersham). Hybridization was performed overnight at 55uC in Church Buffer (0.25 M Sodium Phosphate, pH 7.2; 1 mM EDTA; 6.6% SDS; 1% BSA). The 5S rDNA probe was a 5S LT1 PCR fragment internally labeled with a[32P]-dCTP by random priming. The membrane was washed twice 5 min. with 26 SSC, 0.1% SDS and twice 20 min. with 0.16 SSC, 0.1% SDS; signal was detected by phosphoimaging. PCR analysis of cytosine methylation used 100 ng aliquots of the above digests compared to undigested DNA controls. PCR was performed for 25 cycles using Platinum Taq (Invitrogen) and primers RTPCR5S1 and 5SUNIV2 described in Vaillant et al. (2006) [52]. Fluorescent in situ hybridization and Imaging The 5S rDNA gene probe was labeled with biotin-16-dUTP by PCR as previously described [7]. Meristem nuclei were processed for DNA fluorescence in situ hybridization (FISH) as previously described [81], using 50% formamide and 26 SSC for the hybridization steps. Post-hybridization washes were performed in 50% formamide and 0.16 SSC at 42uC. Biotin-labeled probes were detected using goat anti-biotin conjugated with avidin (1:200, Vector Laboratories) followed by streptavidin-Alexa 546 (1:200, Molecular Probes). Nuclear DNA was counterstained with DAPI in Prolong antifade medium (Invitrogen). Preparations were inspected with a Nikon Eclipse E800i epifluorescence microscope equipped with a Photometrics Coolsnap ES Mono digital camera. Images were acquired using Phylum software, and pseudocolored and merged in Adobe Photoshop 7. Supporting Information Figure S1 Evidence for 5S rRNA degradation in small RNA datasets: A) Alignment of abundant small RNA matches to 5S rRNA genes, illustrated here using a generic 5S rRNA secondary structure and based on sequencing data obtained by Rajagopalan et al. (2006) and Kasschau et al. (2007). B) Identification of likely 5S rDNA degradation products. Sequence datasets were queried for exact matches to two 16-bp interior sequences (yellow boxes) 5S rDNA Silencing by siRNAs proximate to the 59 or 39 ends of major 5S rRNA transcripts. The most frequently obtained reads have 59 ends that correspond to the 5S rRNA 59 terminus, or 39 ends that correspond to the 5S rRNA 39 terminus. Found at: doi:10.1371/journal.pone.0005932.s001 (9.25 MB TIF) Figure S2 Maps of 5S rDNA-derived small RNA from other tissues: A) Map of inflorescence small RNAs matching the 5S rDNA unit repeat, based on analysis of datasets from Rajagopalan et al. (2006) and Kasschau et al. (2007). Small RNA 59-end positions are indicated on the x-axis, with sequencing reads tallied on the y-axis. Upward bars are matches to the forward strand; downward bars represent reverse strand matches. Read tallies are stacked in 10-bp bins, with size-class indicated by color. The diagram at bottom indicates the 5S rRNA gene (thick black arrow), with flanking areas being intergenic spacers. B) Same diagram as panel A, but for seedling datasets from Rajagopalan et al. (2006) and Kasschau et al. (2007). A spike in small RNAs corresponding to the 5S rRNA 59 terminus is apparent in both inflorescence and seedling maps (short arrows), in addition to the 5S rRNA 39 terminus spike (*) and IGS siRNA cluster (**) identified in Figure 1A. Found at: doi:10.1371/journal.pone.0005932.s002 (10.48 MB TIF) Figure S3 DNA methylation analysis of out-crossed nrpd1 and ddm1: Southern blot comparison of (A) Hpa II and (B) Alu I- digested genomic DNA isolated from inflorescences of wild type (WT), homozygous nrpd1 (2/2), heterozygous nrpd1 (+/2), homozygous ddm1 (2/2), and heterozygous ddm1 (+/2). Hpa II tests for cytosine methylation in the symmetric CG and/or CHG contexts, while Alu I tests for asymmetric methylation. The probe corresponds to 5S LT1, as was used in Figures 2E and 3A. Found at: doi:10.1371/journal.pone.0005932.s003 (8.95 MB TIF) Figure S4 Small RNA blot analysis of ddm1-containing double mutants: Complete siR1003 hybridization result shown only truncated in Figure 2C. Found at: doi:10.1371/journal.pone.0005932.s004 (2.85 MB TIF) Figure S5 5S LT1 transcripts not detected in rdr6: RNA samples from inflorescences of wild type (WT), ddm1 and rdr6 were analyzed by one-step RT-PCR. Reverse transcription was performed using R primer, and PCR performed using F and R primers (Figure 1A, diagram). Control reactions were performed using ACT2 primers. RT enzyme was omitted from duplicate 5S LT1 and ACT2 reactions (no RT). Found at: doi:10.1371/journal.pone.0005932.s005 (1.85 MB TIF) Table S1 Statistical analysis of RNA-FISH and DNA-FISH data [MS Word]: A) RNA fluorescent in situ hybridization with the siR1003 probe in wild-type (WT) and ddm1 interphase nuclei (see Figure 2B). Signals restricted to a prominent nucleolar structure devoid of DAPI staining, the nucleolar dot, were classified as ‘‘Nucleolar dot only’’. Signals observed dispersed both in and outside the dot, or only outside the dot, were tallied separately. B) DNA Fluorescent in situ hybridization with the 5S rDNA probe in nuclei from WT and ddm1-containing lines (see Figure 4A). Nuclei were scored for 5S rDNA colocalization with intensely DAPI- stained chromocenters, or a lack thereof. Fisher’s exact test was used to compare WT percentage localization outside chromocen- ters to that of ddm1, nrpd1 ddm1, rdr2 ddm1, and dcl3 ddm1 (WTRX1): all differences were significant (p,0.05). Localization outside chromocenters in ddm1 was then compared to that of nrpd1 ddm1, rdr2 ddm1, and dcl3 ddm1 (WTRX2): in this case, differences with respect to nrpd1 ddm1 and rdr2 ddm1 were significant (P,0.05). PLoS ONE | www.plosone.org 9 June 2009 | Volume 4 | Issue 6 | e5932