Ribominus, as many of you know, is pretty labor intensive and inefficient. We are interested in using DSN normalization in construction of SOLiD WT libraries (total RNA, not just poly(A)). DSN seems easier and more efficient in removing all highly-abundant transcripts (more than just rRNAs). However, there's not that much information about how to build DSN WT libraries with Illumina, let alone SOLiD.
My question is, at what step should we insert the DSN protocol? Since at the end of DSN there is an amplification step, taking the purified cDNA into DSN, then going back to size selection and amplification seems like the best solution.
My concerns are that we won't have the correct amount of material for DSN (80-100 ng of DNA), and that there are some other complications we haven't foreseen. If anyone else has thought about this, or has expertise in SOLiD WT or Illumina DSN, we would greatly appreciate any advice.
EDIT: After consultation, it seems like the proper order is to do the entire SOLiD WT library construction protocol, then do DSN, then follow with another amplification round. It would still be great to hear everyone's thoughts.
My question is, at what step should we insert the DSN protocol? Since at the end of DSN there is an amplification step, taking the purified cDNA into DSN, then going back to size selection and amplification seems like the best solution.
My concerns are that we won't have the correct amount of material for DSN (80-100 ng of DNA), and that there are some other complications we haven't foreseen. If anyone else has thought about this, or has expertise in SOLiD WT or Illumina DSN, we would greatly appreciate any advice.
EDIT: After consultation, it seems like the proper order is to do the entire SOLiD WT library construction protocol, then do DSN, then follow with another amplification round. It would still be great to hear everyone's thoughts.
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