Sample Details

Lenschow Lab

Mouse Ensembl GRCm38.76

Sample Table

Library Prep Details

Total RNA integrity was determined using Agilent Bioanalyzer or 4200 Tapestation. Library preparation was performed with 10ng of total RNA with a Bioanalyzer RIN score greater than 8.0. ds-cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing (Takara-Clontech) per manufacturer's protocol. cDNA was fragmented using a Covaris E220 sonicator using peak incident power 18, duty factor 20%, cycles per burst 50 for 120 seconds. cDNA was blunt ended, had an A base added to the 3' ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12-15 cycles using primers incorporating unique dual index tags. Fragments were sequenced on an Illumina NovaSeq-6000 using paired end reads extending 150 bases.

Alignment Summary

Alignment Summary

Alignment Summary Plots

Column

Total Aligned Fragments

Average Read Length

Alignment Bias and Saturation

Column

EndBias Plot: A plot of the 3 prime and 5 prime bias averaged across all known genes for a given sample. Ideally, the plot should show high and consistent coverage from the 3 prime to the 5 prime ends that appears as a large plateau over the normalized 100bp gene body. High coverage at one end and low coverage at the opposite end indicates end bias during sample and library preparation. Inconsistent spikes in coverage across the gene body with low coverage in between spikes indicates sample degradation or library preparation artifacts that are typical with non-strand displacing random priming library preparation such as the Sigma kit for low input or degraded FFPE samples.

Column

Junction Saturation Plot: A plot of the coverage over known and novel splicing junctions. When the known/novel junctions plateau, the plot indicates that the sensitivity for known/novel junctions has been maximized and continued sequencing would only increase coverage rather than sensitivity. If the plot shows no signs of plateauing for the known/novel junctions, the plot indicates that low and low-medium expressors are likely under-sampled and the abundances measured will not reflect the true distribution of expressed genes or transcripts. Continued sequencing will increase sensitivity and would likely prove beneficial for all downstream analysis such as gene or transcript level differential expression.

Gene Summary

Gene Expression Summary

Gene Biotype Summary

Column

Gene Biotype Table

Gene Biotype Plot

Gene Model Coverage

Column

Column

Gene Correlation

Column

Gene Spearman Correlation Matrix

Column

Gene Pearson Correlation Matrix

Isoform Summary

Isoform Summary

Isoform Correlation

Column

Isoform Spearman Correlation Matrix

Column

Isoform Pearson Correlation Matrix