SUPPORTING INFORMATION
Additional Supporting Information may be found in the online version of this article:
Figure S1. Dot-blot hybridization of amplified mCIP DNA.
Equal amounts of mCIP and input DNA were dot-blotted (twice for each DNA
sample), on membranes and hybridized with 32P-labeled noaCRR1 and mtDNA1,
respectively. Note the significant sequence enrichment of noaCRR1 but not
mtDNA1.
Figure S2. Real-time qPCR assay of methylation enrichment.
Real-time qPCR analysis for testing the enrichment of mCIP DNA over input DNA,
using primers targeting mtDNA2, the 5’ LTR regions of retrotransposons RIRE3 and
noaCRR2, and four Cen8 genes. Cen8.t00118, Cen8.t00222 and Cen8.t00423 are
inactive genes and Cen8.t00647 is an active gene (Yan et al., 2005). The relative fold
enrichment (RFE) level was calculated as described (Yan et al., 2005) and shown as
histogram bars with standard error (n=3), with mtDNA2-1 used as the baseline control
(RFE=1).
Figure S3. Mapping of mCIP sequence reads.
Reads showing alignments of 20 bp with the assembled CentO sequences were
classified as CentO-containing reads. The remaining reads were mapped to the rice
genome using ELAND and SOAP (see Experimental Procedures) by allowing no
more than two mismatches and a minimal alignment length of 25 bp.
Figure S4. DNA methylation within promoter regions.
The size coverage of promoters by methylation peaks was calculated for a total of
46,327 genes, including 12,908 TE-related, 18,478 active and 14,941 predicted genes.
The 5’ ends of these genes were 1 kb away from the adjacent genes. The genes were
divided into five different size groups: <0.5 kb (A), 0.5-2 kb (B), 2-4 kb (C), 4-6 kb
(D), and >6 kb (E). (F) All genes. Proximal promoter: upstream 200 bp; distal
promoter: upstream 201-1000 bp.
Figure S5. Confirmation of DNA methylation by bisulfite sequencing.
Frequency of methylated cytosine is shown as the height of the histogram bars on the
y-axis and the position of individual cytosines in the sequenced portion is shown on
the x-axis. Red, green and blue histogram bars indicate the frequency of methylated
cytosine in the context of CG, CHG and CHH, respectively. The order of the 13
regions (from A to M) is the same as that in Table S2.
Figure S6. Distribution of mCIP-seq read density across three types of genes.
Genes were classified into groups based on the different coverages of gene bodies by
methylation peaks, from <5% to 30%. The vertical solid and dashed lines represent
the transcription start site and gene end, respectively.
Figure S7. Distribution of DNA methylation across three types of genes.
Annotation is as described in Figure 3, except that the y-axis represents the coverage
of each bin by methylation peaks.
Figure S8. Methylation of centromeric DNA sequences associated with the CENH3
and H3 subdomains in three rice Cen5 and Cen8.
(a) Coverage of each defined region or the same set of regions by methylation peaks.
(b) The same regions as in (a), with read density per 10 kb plotted. For each
centromere, the upper panel denotes intergenic regions (including TE-related genes)
and the lower panel denotes all sequences. See the legend of Figure 8 for description
of each CH3 and H3 subdomain.
Table S1. DNA methylation regions associated with the 12 rice chromosomes.
Table S2. PCR primers for bisulfite sequencing to verify the methylation of a
negative (A) and 12 positive (B-M) regions.
Table S3. PCR primers for mCIP-real time qPCR.