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Torin Experiment Summary

This report summarizes an eRibo Pro experiment and analysis performed with the following samples:

Sample group RPF RNA-Seq
Treated RPF_Torin_Rep1, RPF_Torin_Rep2 RNASeq_Torin_Rep1, RNASeq_Torin_Rep2
Untreated RPF_Untreated_Rep1, RPF_Untreated_Rep2 RNASeq_Untreated_Rep1, RNASeq_Untreated_Rep2

For each starting sample, cells were lysed and 1 µg of total RNA was isolated and heat fragmented for RNA-Seq while 9 µg of total RNA was digested with RNase I to isolate ribosome protected RNA footprints (RPFs). Both samples were taken through rRNA depletion and adapter ligation. Ligated RNA was reverse transcribed, libraries were generated via PCR, and then sequenced on a NextSeq 2000.

After sequencing, samples were processed with Eclipsebio's proprietary analysis pipeline (v1). Unique molecular identifiers (UMIs) were pruned from read sequences using umi_tools (v1.1.1). Next 3' adapters were trimmed from reads using cutadapt (v3.2). Reads were then mapped to a custom, curated database of repetitive elements and rRNA sequences. All non-repeat mapped reads were mapped to the genome hg38 using STAR (v2.7.7a) and PCR duplicates were removed using umi_tools (v1.1.1). Gene coverage was calculated using Eclipsebio's proprietary feature counting algorithm and ribosome occupancy was calculated by dividing coverage in the ribosome associated (RPF) library by the RNA-Seq library. Differentially expressed and occupied genes were identified using DESeq2.

Quality Control Metrics

The following table shows important quality control metrics for each sample. Two libraries are made for each sample, one containing ribosome associated transcripts (RPF) and one from the whole transcriptome (RNA-Seq). For each sample ("Sample"), two rows are included that list the metrics for each library. The identity of the library is included in the "Library" 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 reads: this column lists the number of uniquely mapped, deduplicated reads that were used for this analysis.

Clicking on the sample name for the RPF libraries will open up another HTML report, which gives more detailed plots and information regarding that particular sample. Please note that all HTML reports must be stored in the same location on your computer for the link to work.

Sample Library Initial reads % Pass trim % Repetitive elements % Uniquely aligned to the genome % PCR duplicates Final reads

Gene Coverage Range

Pairs of ribosome associated RNA (RPF) and RNA-Seq libraries were prepared from each sample. The plot below shows the distribution of RPM (reads per million) normalized gene counts in each of the libraries.