Qi et al. 26-3-2564C Qi et al., Figure S1 sirna count per 5 kb small RNAs matching the direct strand sirna count per 5 kb small RNAs matching the complementary strand Repeat length (bp) per 5 kb repeats in the chromosome pericentromeric Position (bp) on chlomosome 1
Qi et al. 26-3-2564C Figure S1. Chromosome-wide density analysis of the AGO4-associated small RNAs on Chromosome 1. The non-redundant set of small RNAs was screened against the Arabidopsis genome using WU-BLAST. The density of small RNAs with perfect matches to the direct strand (upper panel) or the complementary strand (middle panel) of Chromosome 1, and the density of repeats (lower panel) on Chromosome 1 were plotted in a 5 Kb sliding window. The position of the pericentromeric region is marked.
Qi et al. 26-3-2564C Qi et al., Figure S2 sirna count per 5 kb small RNAs matching the direct strand sirna count per 5 kb small RNAs matching the complementary strand Repeat length (bp) per 5 kb repeats in the chromosome pericentromeric Position (bp) on chlomosome 2
Qi et al. 26-3-2564C Figure S2. Chromosome-wide density analysis of the AGO4-associated small RNAs on Chromosome 2. The non-redundant set of small RNAs was screened against the Arabidopsis genome using WU-BLAST. The density of small RNAs with perfect matches to the direct strand (upper panel) or the complementary strand (middle panel) of Chromosome 2, and the density of repeats (lower panel) on Chromosome 2 were plotted in a 5 Kb sliding window. The position of the pericentromeric region is marked. 2
Qi et al. 26-3-2564C Qi et al., Figure S3 sirna count per 5 kb small RNAs matching the direct strand sirna count per 5 kb small RNAs matching the complementary strand Repeat length (bp) per 5 kb repeats in the chromosome pericentromeric Position (bp) on chlomosome 3
Qi et al. 26-3-2564C Figure S3. Chromosome-wide density analysis of the AGO4-associated small RNAs on Chromosome 3. The non-redundant set of small RNAs was screened against Arabidopsis genome using WU-BLAST. The density of small RNAs with perfect matches to the direct strand (upper panel) or the complementary strand (middle panel) of Chromosome 3, and the density of repeats (lower panel) on Chromosome 3 were plotted in a 5 Kb sliding window. The position of the pericentromeric region is marked. 3
Qi et al. 26-3-2564C Qi et al., Figure S4 sirna count per 5 kb small RNAs matching the direct strand sirna count per 5 kb small RNAs matching the complementary strand Repeat length (bp) per 5 kb repeats in the chromosome pericentromeric Position (bp) on chlomosome 5
Qi et al. 26-3-2564C Figure S4. Chromosome-wide density analysis of the AGO4-associated small RNAs on Chromosome 5. The non-redundant set of small RNAs was screened against the Arabidopsis genome using WU-BLAST. The density of small RNAs with perfect matches to the direct strand (upper panel) or the complementary strand (middle panel) of Chromosome 5, and the density of repeats (lower panel) on Chromosome 5 were plotted in a 5 Kb sliding window. The position of the pericentromeric region is marked. 4
Qi et al. 26-3-2564C a 6 Qi et al., Figure S5 5 4 3 Total AGO1 AGO4 2 1 b mir159 mir16 mir161 mir164 mir166 mir167 mir168 mir172 mir319 mir39
Qi et al. 26-3-2564C Figure S5 Preferential association of a subset of micrornas with AGO4. a. Data obtained from 454 sequencing were used to quantify levels of individual mirnas, relative to total mirna content, in AGO1 and AGO4 complexes and in total RNA. Percent contribution for a subset of micrornas with preferential association with either AGO1 or AGO4 is shown graphically. b. Northen analysis of mir172, mir163, mir39, and mir173 confirm patterns of AGO1 and AGO4 binding discerned from sequencing. sirnas from AtSN1 and tasirna48 are shown as controls for AGO4 and AGO1-bound species, respectively. 5
La-er Col La-er Col La-er Col La-er Col La-er Col Qi et al. 26-3-2564C Qi et al., Figure S6 a Total RNA Control TAPAGO4 b 3 nt La-er Col dcl3-1 rdr2-1 AtREP2 3 nt 2 nt AtREP2 3 nt 2 nt SIMPLEHAT2 3 nt 2 nt 3 nt SIMPLEHAT2 mir171 2 nt 2 nt c Percentage of methylated cytosine AtREP2 8 6 4 2 CpG CpNpG CpHpH dcl3-1 rdr2-1 4 3 2 1 dcl3-1 rdr2-1 1 8 6 4 2 dcl3-1 rdr2-1 SIMPLEHAT2 Percentage of methylated cytosine 1 8 6 4 2 CpG dcl3-1 rdr2-1 2 15 1 5 CpHpH dcl3-1 rdr2-1
Qi et al. 26-3-2564C Figure S6 Identification of AGO4-regulated loci through small RNA sequencing. a, Northern analysis of small RNAs in total RNA and in RNAs isolated from TAPAGO4 and control purifications were performed using the indicated probes. Radioactive RNAs of known sizes were included as size markers. b, Northern analysis of AtREP1/2 and SIMPLEHAT2 sirnas were performed on RNA from the indicated plants. mir171 serves as a loading control. c, Analysis of CpG (left), CpNpG (center), and CpHpH (right) methylation at ATREP2 and SIMPLEHAT2 loci was carried out by bisulfite sequencing of genomic DNA prepared from the indicated plants. The methylation level is shown by the percentage of methylated cytosine in all sequenced clones. Data in Table S6 were used to generate the histograms. 6
Qi et al. 26-3-2564C Qi et al., Figure S7 Control AGO4 D66A D742A H874A
Qi et al. 26-3-2564C Figure S7. Accumulation of AGO4 wild-type and DDH mutant proteins in transgenic Arabidopsis plants. Total proteins were extracted from pooled samples of ~3 T1 transgenic lines for each constructs. Western blot analysis was performed using anti-myc antibody. The position of myc-ago4 is indicated by the arrow. The lower band is a cross-reacting species seen in all samples. 7
Qi et al. 26-3-2564C Qi et al., Figure S8 Input RNA Control IP AGO4 IP Input RNA Control IP AGO4 IP Substrate 5 cleavage product TAS3 (mir39) AP2 (mir172)
Qi et al. 26-3-2564C Figure S8. AGO4-mediated cleavage of mirna targets. AGO4 complexes or control immunoprecipitates were mixed with labeled, synthetic target RNAs for two AGO-4 associated micrornas, mir39 and mir172 (TAS3 and AP2 mrna, respectively). Cleavage was monitored by electrophoresis of reaction products. 8
Qi et al. 26-3-2564C Qi et al., Figure S9 CpG CpNpG CpHpH 25 25 2 Percentage of methylated cytosine 2 2 1.5 SUP 15 1 15 1 1 5 5.5 4 25 2 3 2 1.5 AtMu1 Percentage of methylated cytosine 2 15 1 1 1 5.5 1 6 3 MEA-ISR Percentage of methylated cytosine 8 6 4 5 4 3 2 25 2 15 1 2 1 5 8 4 8 6 3 6 AtREP2 Percentage of methylated cytosine 4 2 4 2 1 2 clk-st Control#L1 Control#L2 AGO4#L2 AGO4#L3 D66A#L8 D66A#L12 D742A#L1 D742A#L2 H874A#L2 H874A#L3 clk-st Control#L1 Control#L2 AGO4#L2 AGO4#L3 D66A#L8 D66A#L12 D742A#L1 D742A#L2 H874A#L2 H874A#L3 clk-st Control#L1 Control#L2 AGO4#L2 AGO4#L3 D66A#L8 D66A#L12 D742A#L1 D742A#L2 H874A#L2 H874A#L3
Qi et al. 26-3-2564C Figure S9. Analysis of CpG (left), CpNpG (middle), and CpHpH (right) methylation at SUP, AtMu1, MEA-ISR and ATREP2 loci in T2 transgenic lines. Bisulfite sequencing data from two biological replicates were combined. Methylation levels are shown by the percentage of methylated cytosines in all sequenced clones. Data from Table S8 were used to generate the histograms. 9
Control#L1 Control#L2 AGO4#L2 AGO4#L3 D66A#L8 D66A#L12 D742A#L1 D742A#L2 H874A#L2 H874A#L3 Qi et al. 26-3-2564C Qi et al., Figure S1 clk-st 3 nt MEA-ISR 2 nt Relative levels to clk-st.1.7.6.43 1..16.23.24.14.14.16 3 nt 2 nt Relative levels to clk-st.22.21.15 1.12 1.32.36.34.32.29.29.32 3 nt 2 nt AtREP2 AtSN1 Relative levels to clk-st.2.2.2.47.77.2.27.2.2.21.29 3 nt 2 nt mir171
Qi et al. 26-3-2564C Figure S1. Northern blot analysis of sirnas derived from MEA-ISR, AtSN1, and ATREP2 in RNAs prepared from the indicated T2 plants. mir171 was used as a loading control. The sirna signals were normalized relative to mir171 and the relative levels were calculated by comparison to those in clk-st (see text, arbitrarily set to 1). 1