The study identifies a class of short RNAs, termed Transcription Start Site-associated RNAs (TSSa-RNAs), in mammalian cells that challenge the conventional unidirectional transcription initiation by RNA polymerase II (RNAPII). TSSa-RNAs are found to be transcribed divergently from active protein-coding gene promoters, with sense and antisense peaks located upstream and downstream of the transcription start site (TSS), respectively. Both bound RNAPII and H3K4-trimethylated histones are present at both sense and antisense TSSa-RNA positions, while H3K79-dimethylated histones, a signature of elongating RNAPII, are unidirectionally downstream of TSSs. These findings suggest that divergent transcription over short distances is common at active promoters and may help maintain a poised state for subsequent regulation. The study also reveals that TSSa-RNAs are associated with genes expressed at varying levels, with a higher bias towards genes with higher expression levels. Additionally, the distribution of TSSa-RNAs around TSSs shows a correlation with chromatin structure, indicating that divergent transcription initiation is widespread but productive elongation by RNAPII occurs primarily unidirectionally downstream of TSSs.The study identifies a class of short RNAs, termed Transcription Start Site-associated RNAs (TSSa-RNAs), in mammalian cells that challenge the conventional unidirectional transcription initiation by RNA polymerase II (RNAPII). TSSa-RNAs are found to be transcribed divergently from active protein-coding gene promoters, with sense and antisense peaks located upstream and downstream of the transcription start site (TSS), respectively. Both bound RNAPII and H3K4-trimethylated histones are present at both sense and antisense TSSa-RNA positions, while H3K79-dimethylated histones, a signature of elongating RNAPII, are unidirectionally downstream of TSSs. These findings suggest that divergent transcription over short distances is common at active promoters and may help maintain a poised state for subsequent regulation. The study also reveals that TSSa-RNAs are associated with genes expressed at varying levels, with a higher bias towards genes with higher expression levels. Additionally, the distribution of TSSa-RNAs around TSSs shows a correlation with chromatin structure, indicating that divergent transcription initiation is widespread but productive elongation by RNAPII occurs primarily unidirectionally downstream of TSSs.