Hi all,
I'm trying to perform guide-seq protocol (https://www.protocols.io/view/guide-...xygxmwool8j/v1) using 293T cells.
The library prep steps are intuitive: gDNA extraction, fragmentation, end-repair, TA adapter ligation, PCR1, and PCR2. The thing that puzzled me is that I always got very few reads from my experiment (2K-200K reads) from the Mi-seq run. Tapestation for PCR2 product showed a good size range of library DNA (tho the average length of library is ~1kb, a bit big; see pic). So far, I ruled out the possibility of incomplete denaturation (my colleague's samples are pooled together with mine in the same run, he got good amount of reads). What else could be contributing to this? Thank you!
I'm trying to perform guide-seq protocol (https://www.protocols.io/view/guide-...xygxmwool8j/v1) using 293T cells.
The library prep steps are intuitive: gDNA extraction, fragmentation, end-repair, TA adapter ligation, PCR1, and PCR2. The thing that puzzled me is that I always got very few reads from my experiment (2K-200K reads) from the Mi-seq run. Tapestation for PCR2 product showed a good size range of library DNA (tho the average length of library is ~1kb, a bit big; see pic). So far, I ruled out the possibility of incomplete denaturation (my colleague's samples are pooled together with mine in the same run, he got good amount of reads). What else could be contributing to this? Thank you!
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