Table 1.

Genomic techniques for assaying transcriptional initiation.

Technique	Method	Results	References
RNA Pol II ChIP Seq	Chromatin is fragmented, Pol II is immuno- precipitated, and the interacting DNA is sequenced.	Identifies DNA that is bound by RNA Pol II in a non-strand specific fashion at ~200 bp resolution.	[81, 82]
cap analysis of gene expression (CAGE)/ SMORE-seq/ TIF-seq	RNA is treated with 5' cap- specific enzyme tobacco acid pyrophosphatase (TAP) and subjected to cDNA sequencing.	Identify 5' ends from stable capped RNAs at single nucleotide resolution.	[18, 83, 84]
global run-on sequencing (GRO-seq) and GRO-cap	Run on assay which restarts RNA Pol II in vitro in the presence of a labeled nucleotide (BrUTP) in order to purify nascent RNA.	Identify nascent RNAs and post initiation pause sites at ~50 bp resolution. Since transcription is restarted in vitro, it can detect unstable transcripts which would normally be rapidly degraded in vivo.	[9, 85, 86]
precision nuclear run-on and sequencing (PRO-seq)	Run on assay which restarts RNA Pol II in vitro using biotin-labeled ribonucleoside triphosphate analogs. By supplying only 1 of the 4 nucleotides at a time, run-on transcription is limited and resolution is improved over GRO-seq.	Identifies nascent RNAs and post-initiation pause sites at <50 bp resolution. Since in GRO-seq type assays transcription is restarted in- vitro, it can detect unstable transcripts which would normally be rapidly degraded in vivo.	[87]
native elongating transcript sequencing (NET-seq)	RNA Pol II associated RNA is purified, and the associated RNA is sequenced.	Identifies nascent RNA at single nucleotide resolution. When combined with CTD phosphorylation specific immunoprecipitation, different populations of RNA can be identified based on the modification status of their transcribing RNA Pol II.	[60, 73, 88]
RNA-seq combined with inhibition of RNA degradation pathways	Different components of various RNA degradation pathways are inhibited (such as exosome components) to enable the isolation of unstable RNAs.	Identifies RNAs regardless of stability. Resolution is variable depending on the RNA seq method employed.	[6]