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  • strand-specific cDNA

    i know that the question is stupid, but i am not sure that i am understanding it.
    i dont see the problem of recognize the sense RNA from double strand cDNA.
    isnt it the one with polyA?
    or,
    i dont understand what is strand-specific cDNA ?

    thanks

  • #2
    Sure, but many of the sequence reads will not have the polyA part in them. So one is not certain whether they derive from sense transcripts, or anti-sense.

    That said, I don't know that strand specificity is essential for de novo transcriptome derivation. Helpful, perhaps, but not essential.

    --
    Phillip

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    • #3
      ok, but..
      why many of the sequence reads will not have the polyA part in them?
      all mRNA molecule have polyA, that is how we can make cDNA.
      we reverse transcrpitase by polyT.
      so when is the level that part of the mRNA/cDNA loosing their polyA tail?

      thanks again..

      Comment


      • #4
        The vast majority of RNAseq protocols use fragmented RNA or cDNA for library construction. This is because there are limits on the length of PCR products that can be generated in a PCR reaction. Or rather, I should write, short PCR products are more robustly amplified than long ones.

        Also, even if you see that polyA tail, you are still inferring that is the transcribed strand. Whereas you can use strand-specific methods to assay which strand it is. Get it? One is a presumption you are making (whether for good reason or not), the other is an observation.

        --
        Phillip

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        • #5
          ok, thank you phillip, i think i got it.

          so if i undersood tou right.
          when converting mRNA to cDNA, i can get the full length of the mRNA as cDNA.
          but after, when i want to create library(probably for PCR amplification) i have to break the cDNA because DNA polymerase has limit around 3K bases.

          so, when i want to amplificate the library that i just broke to fragments, i must use random primers and not just polyT?

          thanks

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          • #6
            Okay, we are starting to get into the details of RNAseq here. But most commonly RNAseq library protocols follow these steps:

            (1) Ribodepletion (removing most of the ribosomal RNA that composes the majority of all total RNA preps.)
            (2) RNA fragmentation. For Illumina this fragmentation will generate a peak around a few hundred nucleotides.
            (3) First and second strand cDNA synthesis. Because of the RNA fragmentation step, the majority of cDNAs will be in the target range.
            (4) ds cDNA end polishing, adapter ligation

            After you ligate the adapters to the cDNA, then you have your library of amplicons. These are usually subjected to other manipulations, but eventually end up being diluted to low titres and amplified by cluster or emulsion PCR to create ~ 1um splotches of area on a flow cell surface with thousands or millions of copies of the same amplicon. Sequencing proceeds from a priming site in one of the adapters.

            Why a few hundred bases instead of something close to the polymerase size limit?

            First, your read length is only 50-100 nt or so. Reading from the very ends of full length cDNAs will only interrogate a percentage of your entire transcriptome. To read the entirety you either need very long read lengths, or you need to fragment your transcripts prior to sequencing them. Second, PCR is much more efficient at replicating smaller amplicons than larger ones. So by going shorter you get a signal boost for your template clusters or beads.

            --
            Phillip

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            • #7
              thank you Phillip!
              that was helpful!

              Comment


              • #8
                Does any one know the difference between T7 RNA polymerase based ivt to dUTP second strand synthesis?

                thanks

                Comment


                • #9
                  Originally posted by papori View Post
                  Does any one know the difference between T7 RNA polymerase based ivt to dUTP second strand synthesis?

                  thanks
                  T7 IVT is an linear amplifcation step. Basically, double stranded cDNA is created using an oligo dT that also includes the T7 promoter sequence. T7 polymerase is then used to generate cRNA as it only transcribes from DNA downstream of the T7 sequence. It generates amplified strand specific cRNA.
                  Typically it's used for expression microarrays that require large amounts of material for hybridisiation.

                  The dUTP RNA seq method uses an oligo dT to prime mRNA, then deoxyuridine triphosphate instead of deoxythymidine triphosphate is used in the synthesis of the second strand of cDNA. cDNA is then fragmented and a library prepared (fragmentation, end repair and ligation). Before the final PCR amplification step, an enzyme (Uracil-N-Glycosylase) is used to cut the second strand at all the uracil bases, so only the first strand can be sequenced - maintaining the strand specificity.

                  Comment


                  • #10
                    Originally posted by TonyBrooks View Post
                    T7 IVT is an linear amplifcation step. Basically, double stranded cDNA is created using an oligo dT that also includes the T7 promoter sequence. T7 polymerase is then used to generate cRNA as it only transcribes from DNA downstream of the T7 sequence. It generates amplified strand specific cRNA.
                    Typically it's used for expression microarrays that require large amounts of material for hybridisiation.
                    Though not an exponential amplification, T7 RNA polymerase does produce quite a few copies of RNA from each DNA strand (by displacement of the nascent strands, I presume).

                    Affy had a protocol (the 2 cycle amplification method) that converted the RNA produced by T7 back into cDNA followed by another cycle of T7 amplification. That gave near-exponential amplification.

                    --
                    Phillip

                    Comment


                    • #11
                      I joined that thread because the first post was a "stupid" question and those are often the most interesting ones.. Thanks Phillip and Tony for the details and explanations.

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                      • #12
                        Thanks TonyBrooks & phillip
                        this is great explanation!

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                        • #13
                          what is the reason to the fact that when we are talking about "strand specific" dUTP is better than 7T IVT?
                          only the linear vs. exponential amplification?

                          Comment


                          • #14
                            T7 IVT is not a library prep method. It is a way of amplifying poly A+ RNA. It produces single stranded cRNA (although it does keep it's strandedness as it's only amplified from one strand). It's mostly used in microarrays where the input needs to be single stranded material.

                            The "dUTP" method is a library prep method. The only amplification is at the end of the protocol when the sample is enriched for adapter ligated material by PCR. Before this step, the second strand (marked with uracil) is degraded to only the first strand is enriched by PCR.

                            Amplification is a red herring here. You can't really compare dUTP and T7 IVT as they are predominantly used for different technologies. Although both methods retain strandedness, it's not an issue with T7 IVT as the microarray will only have probes where the strand of the mRNA is known. Strandedness is much more important in sequencing as you want to know which strand a read has come from to identify novel transcripts.

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                            • #15
                              Maybe i just didnt understand what i read from this paper:
                              Development and applications of single-cell transcriptome analysis
                              Fuchou Tang1,2, Kaiqin Lao3 & M Azim Surani1

                              Combined with the existing strand-specific cDNA library preparation strategies, such as T7 RNA polymerase–based in vitro transcription and dUTP second-strand synthesis strategies it would be possible solve the stranded-ness of mRNAs is lost in the library construction, which prevents discrimination between sense and antisense transcripts from the same locus.

                              isnt it said that combined with both will solve the stranded-ness of mRNAs??
                              Can you explain that?

                              Thanks,
                              pap

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