Additional file 1:

Position-effect variegation (PEV) analysis of male flies of small RNA and RNA Pol II mutations. The trans heterozygote of RNA Pol II and small RNA pathway mutations showed very strong suppression of In(1)w [m4h] PEV compared with control and single heterozygote mutants. All the male flies were of same age (4 days after eclosion).

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Additional file 2:

Immunofluorescence analysis of polytene chromosomes in RNA Pol II and small RNA pathway trans-heterozygote mutants. H3K9me2 modification is strongly reduced in trans-heterozygotes compared with the control. The FITC (green) channel shows H3K9me2 antibody signal while the Texas red shows Sxl antibody signal. Representative images from five different experiments (approx 50 pairs of nuclei) have been examined. The genotypes of each polytene nucleus has been indicated.

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Additional file 3:

Western blot analysis of heterochromatin protein-1 (HP1) in small RNA and RNA Pol II trans-heterozygote mutants. Western blot analysis with HP1 and tubulin (loading control) antibodies on adult carcasses of the indicated genotypes. No significant upregulation of HP-1 was observed in mutants compared with wild type. The standard error bars were calculated from three different experiments.

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Additional file 4:

Western blot analysis to check the specificity of the dcr-2 antibody. The western blot analysis performed on third instar larvae shows the absence of the specific band at ~200 kDa in dcr-2 (L811fsX).

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Additional file 5:

Amino acid sequence of RNA Pol II second largest subunit and dAgo-2. The consensus heterochromatin protein-1 binding pentapeptide sequence (PxVxV) is highlighted in bold letters.

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Additional file 6:

Eye pigment analysis of dcr-1(Q1147X) and RNA Pol II 140(A5) in w [m4h] background. Trans-heterozygotes of dcr-1(Q1147X) and RNA Pol II 140(A5) didn't affect position-effect variegation. Three independent replicas were performed. Standard error is shown. The genotypes of male flies are indicated.

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Additional file 7:

Role of miRNA machinery in heterochromatin formation. Immunofluorescence analysis of polytene chromosomes using H3K9me2 (FITC) and Sxl (Tx red) antibodies on the noted genotypes.

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Additional file 8:

Co-localization of AGO1 and RNA Pol II (8WG16) on polytene chromosomes. The arrows indicate sites of co-localization between AGO1 and RNA Pol II. Canton S wild type third instar larvae were used.

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Additional file 9:

Analysis of AGO1 localization. The upper panels show AGO1 and Sex lethal proteins on female polytene chromosomes. The lower panel is the control experiment demonstrating the specificity of the antibody using AGO1 specific blocking peptide. Sxl is the internal control and it is unaffected by AGO1 blocking peptide.

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Additional file 10:

Effect of Taf-1 (TATA Box Associated factor 1) on position-effect variegation using In(1)w [m4h] male flies. All male flies were of the same age (4 days after eclosion).

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Additional file 11:

Effect of RNA Pol II and RNA silencing machinery on white-IR post transcriptional gene silencing (PTGS). Trans-heterozygotes of RNA Pol II 140 and small RNA mutants don't affect w-IR PTGS. The genotypes of male flies are indicated.

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Additional file 12:

Effect of RNA Pol II 140(A5) and small RNA silencing machinery (hls [125]) on Adh-white (1 copy) and white-Adh (2 copies) female flies. All flies were observed 2 h after eclosion.

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