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Hi!
I want to sequence the 3'-end of all transcripts of a cell using the Nextera kit.
The tagmentation rxn is done on full-lenght ds cDNA and since the oligo-dT used in the RT is biotinylated, I then expect to capture all the 3'-ends of the transcript using Dynabeads (and get rid of the rest).
What is not clear to me is what the transposase is doing at the end of the transcript. Adding one adaptor? None?
I expect the fragment to look like this after tagmentation:
5’NNNNNNNNNNNNNNNNNNN--------------------------------AAAAAAAAAAACCCCCCCCCCCCCC-3’
3’ NNNNNNNNNNN ------------------------AAAAAAAAAAACCCCCCCCCCCCCC-biot 5’
where: N= sequence loaded on the transposase and attached to the target DNA
A=poly-A tail
C=custom adaptors
If I then follow the Nextera protocol and do gap filling+PCR I get nothing...only adaptor dimers (i5-i7).
Does anybody have experience with tagmentation of the 3'end of transcripts? I can't find any useful info on the web.
Any help is appreciated! Thanks in advance!
I want to sequence the 3'-end of all transcripts of a cell using the Nextera kit.
The tagmentation rxn is done on full-lenght ds cDNA and since the oligo-dT used in the RT is biotinylated, I then expect to capture all the 3'-ends of the transcript using Dynabeads (and get rid of the rest).
What is not clear to me is what the transposase is doing at the end of the transcript. Adding one adaptor? None?
I expect the fragment to look like this after tagmentation:
5’NNNNNNNNNNNNNNNNNNN--------------------------------AAAAAAAAAAACCCCCCCCCCCCCC-3’
3’ NNNNNNNNNNN ------------------------AAAAAAAAAAACCCCCCCCCCCCCC-biot 5’
where: N= sequence loaded on the transposase and attached to the target DNA
A=poly-A tail
C=custom adaptors
If I then follow the Nextera protocol and do gap filling+PCR I get nothing...only adaptor dimers (i5-i7).
Does anybody have experience with tagmentation of the 3'end of transcripts? I can't find any useful info on the web.
Any help is appreciated! Thanks in advance!