This interactive report provides an overview of miR-eCLIP +siRNA results, accessible at the different tabs. For each table and plot, the button can be clicked to get more information. The menus in the top right corner of each plot can be used to modify the plots (zoom, hide samples, and save a copy of the plot). This report is optimized for viewing on a desktop and plots have been tested on several browsers but in some cases they may not render correctly, please use an alternate browser if plots do not load.

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Experiment Summary


This report summarizes a miR-eCLIP +siRNA experiment performed with a standard siRNA (APP-01-Unmod), a Dharmacon-modified siRNA (APP-05), and an untransfected control. The samples were processed using Eclipsebio's proprietary miR-eCLIP +siRNA technology to identify off target siRNA binding. Full methods details are available upon a request.

Quality Control Metrics


The following table shows important quality control metrics for each sample. Two libraries are made for each sample, one containing immunoprecipitated (IP) RNA and the other containing input RNA. For each sample ("Sample"), two rows are included that list the metrics for each library. The identity of the library is included in the "IP or Input" column.

The following columns describe the processing of the libraries:

  1.  Initial reads: the number of sequenced reads for each library.
  2.  % Pass trim: reads first underwent adapter trimming to remove both adapters and sequences shorter than 18 nucleotides. The percentage of sequenced reads that passed this trimming step is listed in this column.
  3.  % Repetitive elements: trimmed reads are aligned to Eclipsebio's custom database of repetitive elements. This database includes rRNAs, tRNAs, snoRNAs, and other features. The percentage of trimmed reads that were filtered is listed in the column.
  4.  % Uniquely aligned to the genome: the remaining reads from the repetitive element filtering were aligned to the genome, and the percent of those reads that map uniquely to the genome is listed in this column.
  5.  % PCR duplicates: PCR duplicates were defined as uniquely mapped reads that map to the same read coordinates and have an identical unique molecular identifier (UMI). The percentage of mapped reads that were identified as PCR duplicates is included in this column.
  6.  Final nonchimeric reads: this column lists the number of uniquely mapped, deduplicated, nonchimeric reads that were used for this analysis.
  7.  AGO2 clusters: AGO2 clusters are found using the peak calling tool CLIPper. The clusters are identified from nonchimeric reads in the IP samples and are not input normalized.
  8.  AGO2 peaks: A peak is defined as a cluster with a log2(fold enrichment over the matched input) > 3 and p-value < 0.001.
  9.  Final chimeric reads: The number of chimeric reads that mapped to the genome after trimming the miRNA component of the chimeric read.
  10.  % Chimeras: percentage of chimeric reads that mapped to the genome out of the total number of reads that mapped to the genome (chimeric + nonchimeric)
  11.  Chimeric clusters: chimeric clusters are found using the peak calling tool CLIPper. The clusters are identified from genome mappinf component of chimeric reads in the IP samples and are not input normalized.
Sample IP or Input Initial reads % Pass trim % Repetitive elements % Uniquely aligned to genome % PCR duplicates Final nonchimeric reads AGO2 clusters AGO2 peaks Final chimeric reads % Chimeras Chimeric clusters
APP-01-Unmod_Input_rep1 Input 65,519,521 98.88% 56.54% 64.38% 13.32% 15,711,065
APP-01-Unmod_Input_rep2 Input 58,473,690 98.16% 53.63% 64.21% 12.21% 15,003,949
APP-05_Input_rep1 Input 66,248,677 96.72% 55.16% 66.79% 12.52% 16,786,399
APP-05_Input_rep2 Input 69,135,647 97.19% 58.32% 65.48% 12.91% 15,972,357
Untransfected_Input_rep1 Input 70,911,737 96.55% 56.61% 62.94% 12.93% 16,279,528
Untransfected_Input_rep2 Input 72,531,030 96.54% 57.53% 64.22% 13.69% 16,484,935
APP-05_IP_rep2 IP 90,365,901 78.50% 61.24% 46.68% 15.03% 10,904,904 324,370 7,511 169,216 1.53% 2,915
APP-01-Unmod_IP_rep1 IP 75,065,701 75.63% 53.92% 47.35% 14.12% 10,636,624 343,680 8,196 176,037 1.63% 3,297
APP-01-Unmod_IP_rep2 IP 95,063,987 75.30% 51.28% 50.05% 17.41% 14,416,246 343,680 10,556 244,433 1.67% 4,228
Untransfected_IP_rep1 IP 91,929,733 74.56% 58.08% 46.22% 13.94% 11,430,425 331,791 8,371 137,009 1.18% 2,378
Untransfected_IP_rep2 IP 130,837,039 79.88% 63.68% 45.51% 17.89% 14,185,128 331,791 7,394 259,065 1.79% 4,588
APP-05_IP_rep1 IP 105,519,575 79.85% 62.32% 45.31% 16.77% 11,972,134 324,370 6,913 184,483 1.52% 3,274

This tab summarizes the identified AGO2 peaks from the nonchimeric reads.

AGO2 Peak Counts


AGO2 clusters are found using the peak calling tool CLIPper. The clusters identified in the IP sample are then normalized against a paired input sample. An AGO2 peak is defined as a cluster with log2(fold change) ≥ 3 and p-value ≤ 0.001. The bar plot below shows the number of significantly enriched AGO2 peaks detected in each sample, as well as the number of significantly enriched reproducible AGO2 peaks for each set of replicates. Hover over each bars to see the number of peaks in each set. All plots in this report are interactive, meaning you can zoom in/out, pan to a certain location, and select elements from the legend to remove. Plots can also be downloaded as a separate file. Hover over the top right corner of the plot to see the interactive options.

AGO2 Gene Counts


The bar plot below shows the number of genes containing at least one significantly enriched AGO2 peak per sample, as well as the number of genes containing at least one significantly enriched reproducible AGO2 peak for each set of replicates. Hover over the bars to see the number of genes in each peak set. All plots in this report are interactive, meaning you can zoom in/out, pan to a certain location, and select elements from the legend to remove. Plots can also be downloaded as a separate file. Hover over the top right corner of the plot to see the interactive options.

AGO2 Peak Feature Distribution


The following bar plot depicts the feature distribution of significantly enriched AGO2 peaks in each sample, as well as reproducible AGO2 peaks for each set of replicates. Peaks are annotated according to the following hierarchy: CDS, 5'UTR or 3'UTR, miRNA, intron, and other. Hover over the bars to see the number of peaks and percentage of peaks per feature in each peak set. All plots in this report are interactive, meaning you can zoom in/out, pan to a certain location, and select elements from the legend to remove. Plots can also be downloaded as a separate file. Hover over the top right corner of the plot to see the interactive options.

AGO2 Peak Width Distribution


This boxplot shows the AGO2 peak width distribution of significantly enriched peaks in each sample, as well as reproducible AGO2 peaks for each set of replicates. Hover over the boxplot to see the minimum, lower fence, q1, median, q3, upper fence, and maximum peak widths for each peak set. All plots in this report are interactive, meaning you can zoom in/out, pan to a certain location, and select elements from the legend to remove. Plots can also be downloaded as a separate file. Hover over the top right corner of the plot to see the interactive options.