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Forum: RNA Sequencing 09-21-2017, 03:52 PM
Replies: 15
Views: 6,115
Posted By nucacidhunter
Larger mRNAs will be included in final library...

Larger mRNAs will be included in final library but maybe with less representation because cDNA synthesis is primed by randomers. If you are going to compare libraries prepared with the same method,...
Forum: RNA Sequencing 09-21-2017, 05:14 AM
Replies: 15
Views: 6,115
Posted By nucacidhunter
Library insert size is affected by RNA fragment...

Library insert size is affected by RNA fragment size (user controllable) and random primers that can prime any region along RNA fragments and a fragment will be primed from multiple locations. That...
Forum: RNA Sequencing 09-20-2017, 08:35 PM
Replies: 15
Views: 6,115
Posted By nucacidhunter
According to Illumina table the longest insert...

According to Illumina table the longest insert even without fragmentation is 200bp (130-350 range). To enrich for larger fragments you can clean up with low bead ratio (not double size selection) to...
Forum: RNA Sequencing 09-20-2017, 04:32 PM
Replies: 15
Views: 6,115
Posted By nucacidhunter
If you have annealing and extension set for 45s...

If you have annealing and extension set for 45s then 950bp will be upper limit of amplification.

0.55x/0.15x should select your target range but the final library insert will depend on the size...
Forum: RNA Sequencing 09-19-2017, 06:55 PM
Replies: 15
Views: 6,115
Posted By nucacidhunter
If you can generate libraries in desired size...

If you can generate libraries in desired size with TruSeq (anecdotally some people have not been successful to obtain above 250 bp) then qPCR should not be an issue. You just need to take the average...
Forum: RNA Sequencing 09-19-2017, 02:40 PM
Replies: 15
Views: 6,115
Posted By nucacidhunter
Sequencing TruSeq RNA library with 2x150 will...

Sequencing TruSeq RNA library with 2x150 will read into adapters. To maximise useful sequencing output you should use other kits such as NEBNext Ultra II RNAseq which allows preparing libraries with...
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