1. http://bioinfo.na.iac.cnr.it/ALE-HSA21ALE-HSA21 is accessible at: The following pages are a schematic representation of how to navigate through ALE-HSA21 website and to extract information of interest. The “core” of ALE-HSA21 website is made of two main sections: -Coding Genes -Non Coding Genes

2. By clicking here the user can access the “General Table” of all the genes annotated on the human chromosome 21 (HSA21). By clicking here the user can access the “General Table” containing 7 HSA21 genes with 12 novel transcripts identified by our group. For sake of simplicity, for the “Coding Genes” section of ALE-HSA21, only examples about “Annotated Genes” are shown in the next pages. The “Novel Genes” subsection contains the same type of information, and has been developed with the same rationale.

3. 1 This is the “General Table” for “Annotated Genes” in which all currently annotated transcripts of HSA21 genes are listed. This Table summarizes primary info such as gene symbols (in alphabetical order), transcript ID, genomic coordinates, sense of transcription (strand), number of exons and (if any) UniProt ID. The “Gene symbol” ( ), the “ID” ( ), the “Genomic coordinates” and the “UniProt ID” are clickable elements. As shown in the black box, by clicking the “Genomic coordinates” the user is directly linked to an “ad hoc created” Session of UCSC Genome Browser containing RNA-Seq coverage data, ENCODE ChIP-Seq data for transcription factors and Ensembl, RefSeq and UCSC genomic annotations 2 12

4. “HOW TO EXTRACT INFORMATION FOR CODING GENES IN ALE-HSA21” “Gene-specific” page“Transcript-specific” page Both “Gene-specific” and “Transcript-specific” pages contain 3D exon/intron structures. Each element (promoter, exon, intron and 3’ UTR ) is independently clickable, allowing to access the results of the computational predictive analysis of regulatory sequences, performed as described in the Method Section. By clicking the “GENE SYMBOL” of interest the user access the “Gene-specific” page By clicking the “ID” of interest the user access the “Transcript-specific” page 2 1

5. Official gene symbol Gene name Gene description Links to external databases Image legend Interactive 3D transcript models. Each element (,,, ) is independently clickable) On the right panel are schematically described the different areas of this web page 1 3 4 5 6 3 4 5 6 “Gene-specific” page

6. This web page is dedicated to a specific transcript of interest. Here the user can access different type of data, simply by clicking each element of the transcript (promoter, exon, intron or 3’ UTR). 2 3 4 5 6 In the following pages are described in detail, step-by-step, all the information the user can retrieve by clicking on: 3 Promoter (defined as ±1kb from the TSS, transcription start site); 4 Exons; 5 Introns; 6 3’ UTR (untranslated regions); “Transcript-specific” page All information about promoter, exon, intron or 3’ UTR can be accessed both from this “transcript-dedicated” web page and from the “gene-dedicated” web page (previously described in ), simply by clicking on the elements of interest within the 3D model. 1 Note to the user:

7. 3 3a Direct access to the sequence of the promoter in FASTA format. Direct link to a Session of Genome Browser ad hoc created for ALE-HSA21 containing ENCODE ChIP-Seq data for transcription factors falling within the promoter of interest Predictive analysis of Transcription Factors’ (TFs) binding sites within promoters Download FASTA sequence of the promoter (±1kb from the transcription start site) 3b3c3d3e3f Human TFs predicted to bind the specific sequence Orientation of TF’s binding on DNA Predicted TF’s binding site within the sequence Position of the predicted TF’s binding site within the sequence of interest 3b 3c 3d 3e 3f Weight score provided by RSAT (http://rsat.ulb.ac.be)

8. 5a 45 Direct link to the sequence of interest in FASTA format 4a 5a Download the sequence in FASTA format 4a 4b Binding motif recognized by the splicing factor 4c Category of the regulatory sequence (Exonic or intronic splicing enhancher/silencer, ESE/ESS and ISE/ISS) 4d Splicing factor 4e Sequence recognized by the splicing factor and surrounding nucleotides within the analyzed element 4f and 5b 5c 5d 5e 5f Position of the binding motif within the sequence of interest 4b4c4d4e4f 5b5c5d5e5f Predictive analysis of splicing regulatory sequences within exons/introns

9. 6 6b6c6d6e6f Direct link to the sequence of interest in FASTA format. 6a Human miRNA predicted to bind the analyzed sequence miRNA seed Position of the predicted miRNA binding site (miRNA responsive element, MRE) within the sequence of interest Match between miRNA and the sequence of interest. Exact match to positions 2-8 of the mature miRNA (seed + position 8) followed by an 'A’ is indicated as “8mer-1a”; “7mer-m8” indicates an exact match to positions 2-8 of the mature miRNA (the seed + position 8); “7mer-1a” indicates an exact match to positions 2-7 of the mature miRNA (the seed) followed by an 'A’ (from TargetScan, http://www.targetscan.org) 6b 6c 6d 6e 6f miRNA ID from miRBase database (http://www.mirbase.org) Predictive analysis of miRNA binding sites within 3’ UTRs 6a Download FASTA sequence of 3’ UTR

10. Similarly to the “Coding Genes”, this is the “General Table” in which all the transcripts of HSA21 genes currently annotated as “non-protein coding” are listed. The first column indicates the non-coding category. “Gene symbol”, “ID” and “Genomic coordinates” are clickable elements.

11. “Gene-specific” page “Transcript-specific” page Results of the computational predictions are available for the following categories: lincRNAs and pseudogenes  miRNA binding sites predictions within the entire transcript miRNAs and snoRNAs  secondary structure predictions miRNAs  target genes predictions A B C “HOW TO EXTRACT INFORMATION FOR NON CODING GENES IN ALE-HSA21” By clicking the “GENE SYMBOL” of interest the user access the “Gene- specific” page By clicking the “ID” of interest the user access the “Transcript- specific” page

12. A A1 A2 A3 A4A5 Human miRNA predicted to bind the analyzed sequence miRNA seed Position of the predicted miRNA binding site (miRNA responsive element, MRE) within the sequence of interest Match between miRNA and the sequence of interest. Exact match to positions 2-8 of the mature miRNA (seed + position 8) followed by an 'A’ is indicated as “8mer-1a”; “7mer-m8” indicates an exact match to positions 2-8 of the mature miRNA (the seed + position 8); “7mer-1a” indicates an exact match to positions 2-7 of the mature miRNA (the seed) followed by an 'A' A1 A2 A3 A4 A5 miRNA ID from miRBase database (http://www.mirbase.org) Clickable element to access the predictive analysis of MREs Download FASTA sequence of the entire transcript Predictive analysis of miRNA binding sites within lincRNAs and pseudogenes

13. B B1 B2 Secondary structure predictions Mature miRNA sequence B1 B2 Download FASTA Prediction of secondary structure for snoRNAs and miRNAs

14. C C1 C2 C3 C4 Validated target genes of a specific miRNA according to miRWalk database (http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/genetarget.html ) C1 C2 C3 Clickable element to access the predictive target genes C4 Predicted target genes of a specific miRNA according to miRWalk database (http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/predictedmirnagene.html ) Predicted target genes of a specific miRNA according to co-expression data of CoMeTa database (http://cometa.tigem.it) Venn diagrams showing the intersection of the three target genes’ lists Download FASTA Predictive analysis of miRNAs’ target genes