Help

This page will help you if you are confused or unsure about input parameters or results you have obtained from the web-site. Should you have any question or comment regarding this web-site feel free to write to the email address from the original publication.

Input parameters

Gene ID

Our service can recognize different types of input in gene ID field.

The service mainly relies on Ensembl gene annotation. Therefore it will accept Mus musculus Ensembl gene (e.g. ENSMUSG00000024401) or transcript (e.g. ENSMUST00000025263) ID.

Alternatively you can enter official gene symbol (e.g. Tnf, Cxcl2, Zfp36). You can also try to type gene name synonym, e.g. TTP is not an official gene symbol (Zfp36 is the official gene symbol), but it will be still recognized. In such case you will recieve a notification that gene was found by synonym name.

The third posibility is to type in Refseq transcipt accession number, e.g NM_013693.

Additionally if you are not sure in gene name you can type first letters and execute the search. The service will suggest you possible names. You can try this feature by typing, for example "Cxcl".

Please note that the service will have slightly different behaviour depending on whether you are searching gene (as provided by Ensembl gene ID or gene symbol) or transcript (as provided by Ensembl transcript ID or Refseq ID). For example if trascript search executed you will be able to use keywords for selecting zoom region (see Zoom region).

Motif

Along with binding information it is also possible to visualize location of specific sequence motifs. By default "ATTTA" motif will be displayed. You can type any motif of your choice in the motif field. It is only allowed to use A,T,C,G nucleotides, minimal length of motif is 4, maximal length is 10.

Zoom region

Mouse genes and transcripts usually have length of several thousand base pairs. The zoom region options allow to have a closer look to the particular regions of a gene/transcript. There are 3 types of input format in this field:

  • interval start-end, e.g. 100-200 (minimal interval length - 20nt);
  • single numeric value, e.g. 100, which is treated as the length of interval centered around position with maximum cross-links;
  • pair position/interval length (e.g. 1000/100). The value before slash is position of interval center, the value after slash is the interval length. In the example above the 950-1050 interval will be produced;
  • one of the keywords (5UTR, CDS, 3UTR). Since in many cases it is not possible to uniquely assign CDS and UTRs to the gene, those keywords are only functional if you prodive Ensembl transcript ID or Refseq accession number.

The figure with zoom region will appear under the full length figure.

PAR-iCLIP experiment (dataset)

We performed the following PAR-iCLIP experiments

  • "TTP LPS 3 h". IP for TTP (Zfp36) in BMDMs stimulated with LPS for 3 h.
  • "TTP LPS 6 h". IP for TTP (Zfp36) in BMDMs stimulated with LPS for 6 h.
  • "HuR LPS 6 h". IP for HuR (Elavl1) in BMDMs stimulated with LPS for 3 h.

TTP PAR-iCLIP was performed in 3 replicates at each time point. HuR PAR-iCLIP was performed in 2 replicates. Since it was shown that replicates correlate extremely well with each other, the result only of one replicate from each experiment is being displayed.

Results and interpretation

PAR-iCLIP visualization

This section represents the results of chosen PAR-iCLIP experiment. At the upper part of the figure number of crosslinks (position 0 of the read) are shown at each genomic position. The origin of x-axis is placed at the most-upstream TSS (transcription start site) of all transcripts for the gene. At the bottom part of the figure motif locations, detected binding sites and all transcript models for the gene are displayed. Motifs are shown as orange rectangles under the x-axis. If there are significant binding sites detected (see Methods), they will be shown as blue rectangles. Transcript model is represented as lines and rectangles of different color. 5'UTR exons are represented as green rectangles, CDS exons are light blue rectangles, 3'UTR exons are red rectangles and introns are shown as lines.

Sites which we detected with the algorithm will be presented in the table. You can find site's mm9 coordinates, coordinates recalculated to the coordinates origin (TSS coordinates) and the binding score. Binding score is defined as the number of crosslinks within binding site.

LPS expression profile

The figure shows the gene FPKM dynamics during LPS stimulation in WT and TTP-/- BMDMs. Error bars show 95% confidence interval calculated based on 3 replicates. Please refer to differential expression section to check the statistical significance of the difference between genotypes. The table shows the FPKM values at each point during the course LPS-induced inflammation for particular genotype.

LPS induction

The figure presents the gene fold-induction by LPS as compared to untreated (i.e. LPS 0 h). LFC (log2 fold change) and qval (p-value normalized for multiple testing) in the figure and table have been calculated using DESeq2. The LFC induction by LPS is strongly affected by the untreated levels.

Differential expression

The figure shows the LFC (log2 fold change) of gene expression in TTP-/- as compared to WT BMDMs. Thus the positive LFC reflects the elevated expression in TTP-/- cells. Differential expression analysis was performed using DESeq2 software.

RNA stability (half-life)

Rate and time are two related measures of RNA stability. Rate is the slope of decay curve. Time (in min) is calculated from the rate.