Note that the bimodal length distributions of the total small RNAs can be due to our library construction protocol, where shorter and longer fractions are prepared separately

The length distribution per each RNA class demonstrates distinct preferences between these classes.<p><b>Copyright information:</b></p><p>Taken from "Hidden layers of human small RNAs"</p><p>http://www.biomedcentral.com/1471-2164/9/157</p><p>BMC Genomics 2008;9():157-157.</p><p>Published online 10 Apr 2008</p><p>PMCID:PMC2359750.</p><p></p

The histograms show frequencies of transcription start sites (TSS) captured by CAGE

Top and bottom panels show CAGE tag-defined TSS mapping to forward and reverse strand, respectively. The shaded grey box indicates the region with potential for forming a dsRNA by transcription on both strands. The location of the small RNA cluster inside this region is indicated in the central panel as a white box. The numbering of genomic nucleotides is assigned by defining the center of the small RNA cluster as +1.<p><b>Copyright information:</b></p><p>Taken from "Hidden layers of human small RNAs"</p><p>http://www.biomedcentral.com/1471-2164/9/157</p><p>BMC Genomics 2008;9():157-157.</p><p>Published online 10 Apr 2008</p><p>PMCID:PMC2359750.</p><p></p

Northern Blot Analysis of ENOR Transcripts

<p>Mouse whole brain total RNA (10 μg/lane) was used for the analysis except for ENOR2 and ENOR61, where mouse thymus total RNA was used. DNA fragments without any predicted repeated sequences were PCR-amplified from cDNAs in ENORs (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020037#pgen-0020037-st003" target="_blank">Table S3</a>), labeled with ³²P-dCTP (Amersham Biosciences), and then used as probes. RNA size was estimated with an RNA ladder (Invitrogen). ENORs are listed in increasing order based on the estimated length of each region.</p

Clusters of the small RNAs are represented in blue, transcription starts captured by CAGE are represented in pink (supported with single CAGE tag) and red (supported by two or more tags) 45

Antisense transcription to the gene, which is near to the small RNAs, is represented as arrow on the genome representation.<p><b>Copyright information:</b></p><p>Taken from "Hidden layers of human small RNAs"</p><p>http://www.biomedcentral.com/1471-2164/9/157</p><p>BMC Genomics 2008;9():157-157.</p><p>Published online 10 Apr 2008</p><p>PMCID:PMC2359750.</p><p></p

Snapshots of the GEV Showing Transcription

<div><p>(A) The <i>Air</i>/<i>Igf2r</i> locus (Chromosome 17: 12,091,531–12,258,195).</p> <p>(B) The <i>Xist</i>/<i>Tsix</i> locus (X chromosome: 94,835,096–94,888,536).</p> <p>(C) The dystrophin <i>(Dmd)</i> locus (X chromosome: 76,500,000–76,754,601).</p> <p>For the transcripts, cDNA sequences from the RIKEN and public databases are shown, and are colored in brown and purple depending upon their chromosomal strand of origin. Predicted genes from Ensembl, NCBI, and RefSeq databases are shown in gray. CpG islands as defined by the UCSC Genome Browser are shown. Blue circles indicate unspliced, noncoding RIKEN cDNAs with adjunct adenine-rich regions. Red circles indicate RIKEN imprinted cDNA candidates [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020037#pgen-0020037-b038" target="_blank">38</a>].</p></div

Some sequences are alignable with several classes of RNAs and multiple loci on the genome

We assign one sequence to one class based on this flow.<p><b>Copyright information:</b></p><p>Taken from "Hidden layers of human small RNAs"</p><p>http://www.biomedcentral.com/1471-2164/9/157</p><p>BMC Genomics 2008;9():157-157.</p><p>Published online 10 Apr 2008</p><p>PMCID:PMC2359750.</p><p></p

Localization of ENOR Transcripts

<p>qRT-PCR was carried out using total and cytoplasmic RNA from mouse whole brain and the corresponding primer pairs (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020037#pgen-0020037-st003" target="_blank">Table S3</a>). ENORs are listed in increasing order based on the estimated length of each region. Apart from the results shown, we also examined the localization of other mRNAs <i>(β-actin</i> and <i>GAPDH)</i> and additional regions of <i>Rian</i> and other ENORs, and these results were consistent with the rest (unpublished data).</p

Presence of Transcription between Adjacent cDNAs

<p>PCR was carried out with and without reverse transcription (RT[+] and RT[−], respectively) using midbrain total RNA and the corresponding primer pairs (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020037#pgen-0020037-st003" target="_blank">Table S3</a>). PCR using genomic DNA was also carried out as a control. A DNA ladder (Promega; <a href="http://www.promega.com" target="_blank">http://www.promega.com</a>) was used as a size marker. The amplified fragments were confirmed as the expected ones by analyzing digestion pattern using several restriction enzymes. The lower band, observed in the RT(+) lane of the amplified fragment C, seems to be nonspecific, because it was amplified using only the right primer and because it showed a digestion pattern with restriction enzymes quite different from that of the upper band and the band of the genomic DNA (unpublished data).</p

Discovery Pipeline for ENORs

<p>FANTOM and public transcripts were clustered into 37,348 TUs by grouping any two or more transcripts that shared genomic coordinates. Then, the following procedures were applied. (1) Protein-coding TUs were excluded by removing any whose transcripts had an open reading frame of either 150 amino acids or more (RIKEN/MGC cDNAs) or one amino acid or more (non-RIKEN/MGC cDNAs). (2) TUs wholly encompassed within introns of protein-coding TUs were excluded to avoid possible pre-mRNA intronic transcripts. (3) Intron-containing TUs were excluded to select for unspliced transcripts. (4) TUs lacking adjunct adenine-rich regions or containing polyA signals were excluded to select for internally primed transcripts. (5) Remaining UNA TUs that mapped within 100 Kb of one another on the mouse genome (mm5) were clustered together, provided they did not overlap the genomic coordinates of a protein-coding TU/NCBI RefSeq/Ensembl gene model with a CDS of 150 amino acids or more or a noncoding TU with a polyA signal within 100 bp of the 3′ end and without an adjunct adenine-rich region. (6) Reliably expressed UNA TU clusters were selected by identifying those with at least ten supporting ESTs. (7) Selected UNA TU clusters were then manually screened and separated based upon evidence of possible internal transcription state sites (based upon CpG islands, CAGE tags, and EST clusters), resulting in the identification of 66 ENORs.</p

qRT-PCR Analysis

<p>Analysis of (A) <i>Air,</i> (B) ENOR28, and (C) ENOR31 loci. Above in each panel, screen shots of the GEV featuring the loci around <i>Air,</i> ENOR28, and ENOR31 are shown. The orange bars indicate the regions for <i>Air,</i> ENOR28, and ENOR31. cDNA sequences from the RIKEN and public databases are shown. Sequences mapped on the plus strand and minus strand are brown and purple, respectively. Predicted genes from Ensembl, NCBI, and RefSeq databases are shown in gray. For RIKEN imprinted transcripts, imprinted cDNA candidates identified previously [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020037#pgen-0020037-b038" target="_blank">38</a>] are shown. CpG islands as defined by the UCSC Genome Browser are shown. Positions of primer pairs are marked by small vertical arrows. Below in each panel, qRT-PCR results for midbrain, hippocampus, thalamus, striatum, and testis using the corresponding primer pairs are shown.</p