Hi all,
I don't have a ton of experience with library preps, so I'm having trouble figuring out what the issue is/what to do next in this situation. I used the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina to generate cDNA libraries from RNA samples. When I piloted the protocol, my libraries came out looking good, but when I ran my actual samples recently they were super inconsistent - some ok, but most of them having really low yields (low library peak on Bioanalyzer). The library peak is expected at ~300bp. Most of them have a bump in this area, but it's small. There doesn't seem to be an issue of adapter/dimer or primer peaks.
There also doesn't seem to be a relationship between input RNA concentration and library success, and all the input RNA samples looked good on Bioanalyzer.
I'm not sure to what extent this is expected and/or if any of the bad samples are usable.
I've attached three example Bioanalyzer traces - the top one is a good library, the bottom left is a low yield (potentially usable?) library, and the bottom right is a very low yield library. Apologies - the x-axis is showing seconds instead of base pairs, but I labeled the library peaks.
If either of the low yield library examples are usable, is it still an issue that there are inconsistencies with the libraries?
And does anyone have any idea what could have caused this?
Happy to provide more info if that helps.
Thanks!
EDIT 10/2023: Just wanted to provide an update for anyone who may run into similar issues. I don't think it was a problem with the beads/ethanol carryover - I tried letting them dry a little more, but it didn't seem to have much of an impact. In the end, I was able to produce consistently usable libraries by increasing the RNA input and/or adding 1 or 2 PCR cycles at the final library amplification step. The added PCR cycles made a bigger difference for library yield. For reference, I started out using 250ng total RNA input (from mouse brain single tissue punches) + 12 final PCR amplification cycles, but, after piloting, I think a better starting point would be 300-350ng input RNA and 13 PCR cycles (of course, this may differ dramatically based on your input sample type).
I don't have a ton of experience with library preps, so I'm having trouble figuring out what the issue is/what to do next in this situation. I used the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina to generate cDNA libraries from RNA samples. When I piloted the protocol, my libraries came out looking good, but when I ran my actual samples recently they were super inconsistent - some ok, but most of them having really low yields (low library peak on Bioanalyzer). The library peak is expected at ~300bp. Most of them have a bump in this area, but it's small. There doesn't seem to be an issue of adapter/dimer or primer peaks.
There also doesn't seem to be a relationship between input RNA concentration and library success, and all the input RNA samples looked good on Bioanalyzer.
I'm not sure to what extent this is expected and/or if any of the bad samples are usable.
I've attached three example Bioanalyzer traces - the top one is a good library, the bottom left is a low yield (potentially usable?) library, and the bottom right is a very low yield library. Apologies - the x-axis is showing seconds instead of base pairs, but I labeled the library peaks.
If either of the low yield library examples are usable, is it still an issue that there are inconsistencies with the libraries?
And does anyone have any idea what could have caused this?
Happy to provide more info if that helps.
Thanks!
EDIT 10/2023: Just wanted to provide an update for anyone who may run into similar issues. I don't think it was a problem with the beads/ethanol carryover - I tried letting them dry a little more, but it didn't seem to have much of an impact. In the end, I was able to produce consistently usable libraries by increasing the RNA input and/or adding 1 or 2 PCR cycles at the final library amplification step. The added PCR cycles made a bigger difference for library yield. For reference, I started out using 250ng total RNA input (from mouse brain single tissue punches) + 12 final PCR amplification cycles, but, after piloting, I think a better starting point would be 300-350ng input RNA and 13 PCR cycles (of course, this may differ dramatically based on your input sample type).
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