tagRNA-seq - analysis of the transcriptional organization of a bacterium
tagRNA-seq is a modified RNA-seq method which is based on the differential labeling of 5' RNA ends. It enables to discriminate primary from processed 5' RNA ends. The method is described in detail by Innocenti, N. et al. 2015. RNA (New York, N.Y.). 21, 5 (May 2015), 1018–30.
It is based on specific ligation of distinct sequence tags to processed start sites (PSS) and transcription start sites (TSS), thereby gaining insight into regulatory processes at transcriptional and post-transcriptional levels.
We offer two variants of the method, i) with focus on identification of TSS and PSS, and ii) whole transcriptome sequencing including information on TSS and PSS. The two methods are outlined in the schemas below.
A) Original tagRNA-seq method:
B) Wohle transcriptome sequencing including targeting of TSS and PSS
The following steps are carried out during tagRNA-seq cDNA library preparation:
- Removal of rRNA molecules with rRNA depletion kit (e.g. Ribo-Zero bacteria)
- Ligation of tagged RNA adapter to 5' monphosphorylated RNA (labeling PSS)
- Enzymatic conversion of 5' triphosphate (5'PPP) to 5' monophosphate (5'P)
- Ligation of second RNA adapter carrying sequence tag2 to the formed 5'P of primary transcripts (labeling of TSS)
- cDNA library preparation using our random or small RNA protocol
Advantage of method
With dRNA-seq and Cappable-seq not all transcripts can be detected in a single experiment as 5' monphosphorylated RNA is either degraded (dRNA-seq) or removed (Cappable-seq), and thus information on post-transcriptional events is lost
tagRNA-seq represents a comprehensive all in one method for the qualitative and quantitative analysis of the transcriptional organization of a bacterium.
Mapping of sequence reads obtained from tagRNA-seq cDNA library: TSSs upper panel, PSSs lower panel.
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