Hi,
My aim was to use Term-Seq to map RNA 3' ends across the genome, and then standard RNASeq to measure the drop in depth across these 3' sites as a measure of transcription read-through at terminators
For the Term-Seq library prep:
- RNA adapter is ligated to RNA 3' ends
- Reverse transcription
- Sequencing (read passes through RNA 3' end)
As far as I know, the RNASeq was done with conventional Illumina paired end, stranded methods
I am finding that the 3' locations identified by Term-Seq are consistently ~38 bp downstream of apparent the RNASeq 3' locations. This is approximately half the Term-Seq read length (75 bp), not sure if that's a coincidence. RNASeq is PE150. I can't think of any reason this should be. I think I've ruled out anything relating to the computational workflow, as I checked the sequence of the original unprocessed reads matches the predicted 3' location for Term-Seq and RNASeq. I can only presume it is something technical in the library prep, although unfortunately as this was done by a company, methodological detail is a bit lacking.
Does anyone know of anything that might cause this?
Any help much appreciated
