Hi Simone,
I've been using your Smart-seq2 protocol to construct over 20 libraries in the last couple of months w/ great results (Arabidopsois root tissue; around few dozens to few hundreds cells, starting w/ ~30pg-3ng total RNA). This week it stopped working. The problem is somewhere in one of the reactions b4 Tagmentation. There is almost no trace for the ~2000bps peak in the BioAnalyzer after pre-amplification reaction. I know this is one of the weird things we, biologist experience and there is usually no straight forward answer but maybe you have any suggestion or noticed that one of the materials is highly sensitive (e.g., freeze and thaw of the KAPA enzyme).
Besides that, less critical issues, what is the % of mapped reads you get and do you get a lot of rRNA reads (I do). Why did you include the VN nts at the end of the poly(dT) primers and did you try primers that do not have these nts.
Another question is why not using lower Ampure beads ratio after the KAPA reaction same as after the XT reaction to get rid of all oligos that are <200bps.
BTW, I've tried to add more of the poly(dT) primers and it seems that less rRNA is present in the final reads, but as I said, currently, nothing works.
Thanks and have a nice weekend,
Guy
I've been using your Smart-seq2 protocol to construct over 20 libraries in the last couple of months w/ great results (Arabidopsois root tissue; around few dozens to few hundreds cells, starting w/ ~30pg-3ng total RNA). This week it stopped working. The problem is somewhere in one of the reactions b4 Tagmentation. There is almost no trace for the ~2000bps peak in the BioAnalyzer after pre-amplification reaction. I know this is one of the weird things we, biologist experience and there is usually no straight forward answer but maybe you have any suggestion or noticed that one of the materials is highly sensitive (e.g., freeze and thaw of the KAPA enzyme).
Besides that, less critical issues, what is the % of mapped reads you get and do you get a lot of rRNA reads (I do). Why did you include the VN nts at the end of the poly(dT) primers and did you try primers that do not have these nts.
Another question is why not using lower Ampure beads ratio after the KAPA reaction same as after the XT reaction to get rid of all oligos that are <200bps.
BTW, I've tried to add more of the poly(dT) primers and it seems that less rRNA is present in the final reads, but as I said, currently, nothing works.
Thanks and have a nice weekend,
Guy
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