The distribution of TSSa-RNAs around TSSs shows divergent transcription. (A) Histogram of the distance from each TSSa-RNA to all associated gene TSSs (4). Counts of TSSa-RNA 5′ positions relative to gene TSSs are binned in 20 nucleotide windows. Red and blue bars represent bins of TSSa-RNAs in the sense and anti-sense orientation with respect to gene transcription, respectively. (B) Percentage of annotated mouse genes with indicated number of associating TSSa-RNAs.

In ES cells, TSSa-RNA associated genes are primarily expressed. (A) ES cell expression data was separated into 4 bins based on Log2 signal intensity levels; off = 1-4, low = 5-8, med = 6-12, and high ≥ 13 (9). Gene counts for each gene expression level bin are shown. (B) The ratio of sense to anti-sense reads in each expression bin.

Transcripts from TSSa-RNA associated regions are primarily 20-90 nts long. (A) Map of the sense TSSa-RNA Rnf12 region. (B) Northern analysis for the Rnf12 sense TSSa-RNA using probe 1 in A. Lane 1 is a 10 bp ladder. Lanes 2-5 are detection controls with 15, and 1.5, 0.75, and 0 fMol of synthetic RNAs (RNA oligo 1a/b in A). Lanes 6-8 are material recovered from the enrichment procedure using DNA oligo 1 in A. Lane 6 is J1 ES cell enriched material (ESC), lane 7 is Hela enriched material (H-), and lane 8 is Hela + 15 fMol synthetic RNA oligos 1a/b in A enriched material (H+). (C) Map of the anti-sense TSSa-RNA Ccdc52 region. (D) Northern analysis for the Ccdc52 anti-sense TSSa-RNA using probe 1 in A. Lanes 1-4 are as lanes 2-5 in B, except using RNA oligo 2 in C. Lanes 5-7 are material recovered from the enrichment procedure using DNA oligo 2. Lane 5 is J1 ES cell enriched material (ESC), lane 6 is Hela enriched material (H-), and lane 7 is Hela + 15 fMol synthetic TSSa-RNA-2 in C enriched material (H+). Bracket marks ESC specific transcripts; * marks background band.

Relationship between TSSa-RNAs and chromatin structure. (A) Percentage of genes associated with indicated chromatin marks. T-test gives p-values < 2.2e^-16 for all marks. (B) Schematic of factor binding site mapping using forward and reverse Chip-seq reads. The midpoint between the forward and reverse reads defines the bound factor location. (C) Chromosomal position vs. enrichment ratio for H3K4me3-modified nucleosomes and RNAPII for a representative gene Mknk2. (D) Metagene profiles for forward (green) and reverse (yellow) reads for ChIP-seq data (first three panels) and TSSa-RNAs from the sense (red) and anti-sense (blue) strand (bottom panel). Panels are aligned at the TSS. The TSS is denoted by the arrow. Black numbers on the profiles define the midpoint between forward and reverse peaks. Red and blue bars above the ChIP-seq profiles represent sense and anti-sense TSSa-RNA peak maxima.