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Design and evaluation of genome-wide libraries for RNA interference screens

Thomas Horn12, Thomas Sandmann13 and Michael Boutros1*

Author Affiliations

1 German Cancer Research Center (DKFZ), Div. of Signaling and Functional Genomics and University of Heidelberg, Department of Cell and Molecular Biology, Faculty of Medicine Mannheim, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany

2 University of Heidelberg, Hartmut Hoffman-Berling International Graduate School for Molecular and Cellular Biology, D-69120 Heidelberg, Germany

3 University of Heidelberg, CellNetworks Cluster of Excellence, D-69120 Heidelberg, Germany

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Genome Biology 2010, 11:R61  doi:10.1186/gb-2010-11-6-r61

Published: 15 June 2010

Additional files

Additional file 1:

Detailed NEXT-RNAi workflow for the (a) design and (b) evaluation of dsRNAs and siRNAs.

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Additional file 2:

NEXT-RNAi predictions of siRNA efficiencies using both the 'rational' and 'weighted' methods for 2,431 siRNAs tested by Huesken et al. [35].

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Additional file 3:

NEXT-RNAi summary HTML page for the design of a genome-wide RNAi library for the Drosophila genome. This page provides information about the number of successful designs (here, about 94% of the 74,907 query-sequences could be covered with long dsRNA designs). The 'Links to HTML results' link to detailed reports (Additional file 4) for each design (the full list of links was cut for this figure). 'Links to result files' directly link to NEXT-RNAi output files, such as the tab-delimited result file (the main output file) summarizing all calculations done in one line per design, a FASTA file only containing the final reagent sequences as well as GFF (generic feature file) and AFF (annotation file format) output files for visualization and direct upload of reagents to a genome browser, respectively. Further, links to the user-input text files and to report files (for example, reports about failed designs) are provided.

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Additional file 4:

Detailed output for a long dsRNA that targets the Drosophila gene csw (FBgn0000382). The box 'dsRNA information' provides information about the primers (for example, sequence, melting temperature, GC content) required for the synthesis. 'Primer pair penalty' is an overall quality score for the primer pair. The lower this score is, the higher is the predicted quality of the primer pair. Further, the full amplicon sequence, its length and location in the genome (in the format chromosome:start..end(orientation)) are presented. The 'Target information' box shows the intended target(s) and transcript(s) as well as other (unintended) targets and transcripts ('NA' means that no target was found). The intended transcripts are those with most siRNA hits (here, all 203 19-nucleotide siRNAs target the 4 isoforms of csw). The intended gene is then defined over the intended transcripts. The 'Reagent quality' box shows the overall number of siRNAs (here 19-nucleotide siRNAs) contained within the long dsRNA sequence, the number of siRNAs that are 'On-target' (the intended target) and those that are 'Off-target' or have 'No-target'. Further quality features computed for this run were the number of conserved miRNA seeds ('mirSeed') in this dsRNA, the number of 'Efficient siRNAs' (here equal to the overall number of siRNAs, since the efficiency cutoff was set to 0), the 'Average efficiency score' (mean efficiency score of all siRNAs contained in the long dsRNA), and the number of 'Low complexity regions' and 'CAN' repeats contained in the long dsRNA. Additionally, the overlap to UTRs (this long dsRNA completely overlaps with annotated UTRs) and the sequence homology to all transcripts (here only to the intended target) were analyzed in this run. The 'Genome Browser' box visualizes the long dsRNA in its genomic context.

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Additional file 5:

Summary statistics of RNAi reagents designed by NEXT-RNAi for different organisms. NEXT-RNAi was used to design RNAi reagents for all annotated transcripts included in the latest available genome release. CAN = CA[ACGT] repeats; UTR = untranslated region; SNP = single nucleotide polymorphism.

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Additional file 6:

Summary statistics for Drosophila and human RNAi libraries re-annotated by NEXT-RNAi. CAN = CA[ACGT] repeats; UTR = untranslated region; SNP = single nucleotide polymorphism.

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Additional file 7:

Raw data for comparison of Drosophila RNAi libraries in Figure 4, including number of genes targeted by each library, number of genes targeted by both the compared libraries and number of genes targeted with independent designs (with no sequence-overlap at all).

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Additional file 8:

Primer sequences and target gene information for the independent long dsRNAs designed against 49 Drosophila phosphatases for the knock-down validation study presented in Figure 5.

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Additional file 9:

RPKM (reads per kilobase gene per million reads) values for 49 Drosophila phosphatases from RNA-sequencing of D.Mel-2 cells and knock-downs measured after RNAi with two independent designs by quantitative RT-PCR (Figure 5; Additional file 10).

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Additional file 10:

Results for knock-down validation of two independent RNAi reagents against 49 Drosophila phosphatases. Target-genes were sorted for the measured mRNA knock-down of design one.

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Additional file 11:

Descriptions and default values of design parameters used for NEXT-RNAi version 1.31.

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