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  • #16
    Originally posted by papori View Post
    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
    OK. I've just read the paper. I think it's a bit confusing here as the paper is predominantly about single cell transcription profiling. In order to do this, you will need to amplify your starting material as there's no way you'll get enough library yield from one cell without amplification.

    In fact, what the paper says is:

    "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 will be possible to recover the strandedness information for single-cell transcriptomes in the near future"

    As I understand, it is implying that you could use T7-IVT to amplify into cRNA then from that create a cDNA library, with uracil incorporated into the second strand. This can then go into library prep, have the second strand degraded and finally PCR amplifed. It might give you enough material so to sequence whilst maintaining strandedness. Although IVT is a very 3' biased amplification, so there are probably going to be issues with this approach.

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    • #17
      so if i understood you well, T7-IVT is only the amplification step because the low amount of RNA in single cell. after use T7-IVT, we will degraded the cRNA.
      then - we will use the dUTP method to create cRNA for the PCR step?

      why do we have to use T7-IVT for the first step?(it not so clear...)
      Double amplification?
      maybe because T7-IVT have less by-products?

      Thanks TonyBrooks!

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      • #18
        Originally posted by papori View Post
        so if i understood you well, T7-IVT is only the amplification step because the low amount of RNA in single cell. after use T7-IVT, we will degraded the cRNA.
        then - we will use the dUTP method to create cRNA for the PCR step?

        why do we have to use T7-IVT for the first step?(it not so clear...)
        Double amplification?
        maybe because T7-IVT have less by-products?
        Yes, you get higher levels of amplification. The T7 step can effectively bump the amount RNA up 100-fold or so -- at the cost of introducing high 3' bias (and probably also other biases.)

        I would avoid it, where possible. I think the main reason it is still used is that the earlier, microarray, techniques frequently made use of it.

        Actually it isn't really the T7 RNA polymerase that is producing the 3' bias, but the oligo dT primer used for first strand synthesis. It is just one of those quick, sloppy methods that "solve" 2 problems at once (1) avoiding most of the signal from ribosomal RNA and (2) amplifying the starting material.


        --
        Phillip

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        • #19
          How Final PCR amplification of first stand cDNA only amplify first strand cDNA

          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.

          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.
          I read many posts about dUTP strand specific RNA-seq, nobody explained why final PCR amplification step after degradation of second strand of cDNA only amplify the first strand of cDNA. My understanding is PCR always generates double stranded amplicoms. Can anybody answer the question. Thanks in advance.

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          • #20
            It is because you have two different adaptors (annealed in a Y-shape). Without dUTP you will get two different amplicons for each insert due to the Y-adaptors meaning that the first read can start from either end of the insert, but with dUTP you will only amplify one amplicon (which will be double stranded) so all reads from the same transcript will go in the same direction.

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            • #21
              You digest the second strand cDNA before PCR using an enzyme that cleaves wherever there is a uracil base (dUTP is used in place of dTTP in the second strand mastermix). This means when you do the PCR you are only amplifying from the first strand cDNA, hence all reads will from the same strand as the mRNA. The first strand cDNA will be complementary to the mRNA that produced it. The PCR product is still double-stranded

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              • #22
                Appreciate...

                my understanding is:
                when you do PCR with two primers using the first strand cDNA as template, at first cycle it will use one of the primers (complimentary to the first strand cDNA) to generate a reverse complimentary strand that is actually is the second strand cDNA, and the following cycles will use the double strand DNA as template. How does the PCR only amplify the the first strand cDNA????

                Thanks.

                Comment


                • #23
                  Both strands are created in the PCR, but by digesting the second strand you ensure that the P5 adapter is always on the 5' end of the mRNA and P7 is always on the 3' end. This means all reads from the sequencer will be the same sequence as the mRNA that made the library (Illumina read 1 are read P5->P7)

                  Last edited by TonyBrooks; 11-27-2013, 08:12 AM. Reason: including image

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                  • #24
                    The point is that dUTP digestion of second strand is performed before PCR amplification. The following PCR cycles use the normal dNTP, there is no way to use the dUTP digestion to get rid of the second strand??? Thanks.

                    Comment


                    • #25
                      You remove the second strand before PCR. This means the template for the reaction is the first strand only. In an unstranded assay, both strands are used as template - so library is created from both strands.
                      The PCR amplicon is not digested - it is double stranded DNA.
                      Look at the jpg in the link. Sequencing is always in the blue to red direction.

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                      • #26
                        Thanks Tony & Chipper for your beautiful expression... I was also curious about the question asked by chaomeizhang. Thanks chaomeizhang.
                        Last edited by AmitChaurasia; 07-01-2016, 05:08 AM.

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                        • #27
                          Originally posted by Chipper View Post
                          It is because you have two different adaptors (annealed in a Y-shape). Without dUTP you will get two different amplicons for each insert due to the Y-adaptors meaning that the first read can start from either end of the insert, but with dUTP you will only amplify one amplicon (which will be double stranded) so all reads from the same transcript will go in the same direction.
                          Thank you Chipper.

                          I would like to add some note to the explanation.

                          The enrichment PCR does regenerate the DNA strand which was degraded by UDG, but the adaptors on both sides of the strands can not hybridized with the lawn oligos on the flow cell, so these strands will be excluded during the cluster generation and eventually not be sequenced.

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