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Let's say someone wanted to make an RNA library (for the MiSeq or HiSeq) without using RNA-Seq. Is it valid to just remove the ribosomal RNA, make cDNA, and then make a TruSeq library? Would you use the same shearing protocol on the Covaris that you do for genomic DNA even though it's already in smaller pieces?
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It is a valid way to make a library-especially if you do not have sufficent RNA to put into a protocol. The only disadvatage is that you lose stranded-ness.
Yes-I run the same Covaris program for cDNA and DNA. -
Basically, there's no difference at all between an RNASeq library and a transcriptome library, the difference in the two terms is really about how you treat your data. If you need to assemble the data for de novo transcriptome analysis, which it seems you might be doing, you'll want the library to be strand specific as it will greatly aid in assembly.
Aside form that, there's no difference in making a transcriptome library from rRNA depleted cDNA compared to a standard DNA library. Depending on what you're doing, and assuming you use the HiSeq, you may not even need to remove the rRNAs. And depending on which system you do sequence one, you may want to adjust your shearing to give you the best fragment size for the sequencing kit you'll be using (e.g. 750bp fragments for a 600 cycle V3 MiSeq kit versus 4-500 bp for a HiSeq run).Comment
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Essentially this is how some of the libraries were a few years ago...cDNA conversion frist then shearing. It should be noted however, that it was also shown that there is some biases introduced by shearing after making cDNA compared to shearing the RNA first.Comment
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Halfway in, I've realized that cDNA is single-stranded. Does end-repair, a-tailing, and ligating on adapters work on single stranded DNA? Ackkkkk!Comment
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...
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