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  • Originally posted by Simone78 View Post
    Actually not. I tried the opposite, to reduce time for the RT to 15 min, the time that is now recommended for the new Superscript IV (and because I´m tired of waiting 1.5 hours!). Result: the yield was lower and the size slightly lower but not so much, considering is a 80% reduction. However, in my case, concatamers were not visible in any case. You could try and see if this makes things better.
    /Simone
    Thanks! I tried out the 5' biotin TSO, and indeeds it got rid of the concatemer issues I was having with the low input RNA.

    I'm trying hard to coerce as much efficiency and yield out of the protocol as possible, as I'm doing RT on a very low abundance transcript. Looking around different TSO protocols, it seems people have slightly different approach to the cycling condition of the TSO reaction: could you comment on the 72 degree annealing and the subsequent cooling step? some protocol go directly from the annealing to cooling to 42 degree, while the SMART-seq2 protocol specfiically says to cool it on ice. Have you seen what the difference is in terms of yield? How about the length of template annealing step? Do you think increase the time of primer annealing could help with low abundance transcript?

    Comment


    • Originally posted by SunPenguin View Post
      Thanks! I tried out the 5' biotin TSO, and indeeds it got rid of the concatemer issues I was having with the low input RNA.

      I'm trying hard to coerce as much efficiency and yield out of the protocol as possible, as I'm doing RT on a very low abundance transcript. Looking around different TSO protocols, it seems people have slightly different approach to the cycling condition of the TSO reaction: could you comment on the 72 degree annealing and the subsequent cooling step? some protocol go directly from the annealing to cooling to 42 degree, while the SMART-seq2 protocol specfiically says to cool it on ice. Have you seen what the difference is in terms of yield? How about the length of template annealing step? Do you think increase the time of primer annealing could help with low abundance transcript?
      Hi,
      interesting questions. I can tell you that I tried the following:
      -skipped the 3 min @ 72 deg. --> got much less cDNA after RT and PCR. So, apparently this step is important to resolve 2ndary structure in the mRNA.
      - performed the denat at 65 (like in the SSRTIII protocol) or 72 for 2 or 3 min. No difference. Never tried to incubate for a longer time because the longer the RNA is at high T the higher the chance/amount it gets degraded (I think).
      - you can´t add all the reagents and go directly 72--> 42 degrees. The SSRTII is T sensitive and gets inactivated at T>60 degrees.
      Please let me know if you have additional questions, I´ll be happy to help!
      /Simone

      Comment


      • Hi everyone,

        I've notice that lately there has been an extensive discussion on which RTs are best for template switching and cDNA generation from a single cell. I can only say that in my hands SSII was also usually giving the best results keeping in mind that I very much do follow Simone's protocol combining it with some protocols from Regev's Lab . I also have a rather good experience with Maxima H Minus RT and I know several other labs that are using it for single-cell RNA-seq.

        I would be interested to know if anyone has tired using TGIRT III? From the literature these RTs appear to have higher processivity and fidelity than retroviral reverse transcriptases, and from the published papers it seems quite impressive when compared to SSIII. Most interestingly, it seems to be more efficient in template-switching, even if the comparison that has been done so far might not be the most optimal for SSIII. I was just curios if anyone has any experience with this enzyme or any knowledge about it when it comes to RNA-seq.

        Cheers

        Comment


        • Originally posted by Luka View Post

          I would be interested to know if anyone has tired using TGIRT III? From the literature these RTs appear to have higher processivity and fidelity than retroviral reverse transcriptases, and from the published papers it seems quite impressive when compared to SSIII. Most interestingly, it seems to be more efficient in template-switching, even if the comparison that has been done so far might not be the most optimal for SSIII. I was just curios if anyone has any experience with this enzyme or any knowledge about it when it comes to RNA-seq.

          Cheers
          TGIRT III is the commercial name for a new group of enzymes called group II intron reverse transcriptases. I got an aliquot of 3 variants directly from the Lambowitz group and tried it on single cells. I was surprised about the average size of the libraries, 4-5 kb vs 1.5-2 kb with Smart-seq2 protocol! Strand switch reaction works and the processivity of the enzymes is impressive (reaction was only 15 min). That said, it never worked with input below 100 ng. The problem is that TGIRT III bind so tightly to the DNA after RT that you need to treat it with NaOH/HCl or SDS before PCR. If you don´t do any column purification or dilute the RT rxn a lot afterwards, the PCR won´t work because there is too much salt. Doing any purification before PCR is not advisable, but it´s probably the only way to go. I am not sure about this TGIRT III, maybe now it works for much lower inputs than what I used. It looks very expensive, though...
          /Simone

          Comment


          • Originally posted by Simone78 View Post
            Hi,
            interesting questions. I can tell you that I tried the following:
            -skipped the 3 min @ 72 deg. --> got much less cDNA after RT and PCR. So, apparently this step is important to resolve 2ndary structure in the mRNA.
            - performed the denat at 65 (like in the SSRTIII protocol) or 72 for 2 or 3 min. No difference. Never tried to incubate for a longer time because the longer the RNA is at high T the higher the chance/amount it gets degraded (I think).
            - you can´t add all the reagents and go directly 72--> 42 degrees. The SSRTII is T sensitive and gets inactivated at T>60 degrees.
            Please let me know if you have additional questions, I´ll be happy to help!
            /Simone
            Hey Simone,

            Thanks! That's definitely helpful. The some of the protocols I see ask for the RNA template/ primer to be denature at 72, then cool down to 42, then add the rest of the reaction mix (with the enzyme) as quickly as possible while the whole mix is still hovering around 42 degrees. It does all seem to not make that much difference as long as the enzyme is not getting inactivated though.

            Comment


            • Originally posted by Simone78 View Post
              TGIRT III is the commercial name for a new group of enzymes called group II intron reverse transcriptases. I got an aliquot of 3 variants directly from the Lambowitz group and tried it on single cells. I was surprised about the average size of the libraries, 4-5 kb vs 1.5-2 kb with Smart-seq2 protocol! Strand switch reaction works and the processivity of the enzymes is impressive (reaction was only 15 min). That said, it never worked with input below 100 ng. The problem is that TGIRT III bind so tightly to the DNA after RT that you need to treat it with NaOH/HCl or SDS before PCR. If you don´t do any column purification or dilute the RT rxn a lot afterwards, the PCR won´t work because there is too much salt. Doing any purification before PCR is not advisable, but it´s probably the only way to go. I am not sure about this TGIRT III, maybe now it works for much lower inputs than what I used. It looks very expensive, though...
              /Simone
              Hi Simone,
              Glad to see that you already have tested it. The one you got from Lambowitz is the same as the commercial one and I agree it is pricey. I was about to do some tests myself as well as we got some aliquots of the enzyme. I was very impressed when reading the papers how processive is this enzyme. A shift from 1.5-2 kb to 4-5 kb does make me think if at least for some RNA-seq libraries this enzyme should be used. I do single-cell RNA-seq but I frequently run RNA-seq on few hundred cells, never more than 500 so it seems almost perfect for those samples. I presume you have done your tests on purified RNA? You are saying that there is no way to make it work on single cells, but for higher than single cell amounts of RNA it might works as long as one strips the enzyme from DNA. It seems that you have just used the same approach as you did for Tn5, otherwise it just doesn't work?
              Did you get to sequence any of those test libraries, I would also like to understand if a 1.5-2 kb and 4-5 kb library will differ substantially after sequencing? Would you recommend this enzyme for library preps in some particular cases or SSII is good enough?

              Thanks a lot, it's always great to talk to you and learn more about RNA-seq.

              Comment


              • Originally posted by Luka View Post
                Hi Simone,
                Glad to see that you already have tested it. The one you got from Lambowitz is the same as the commercial one and I agree it is pricey. I was about to do some tests myself as well as we got some aliquots of the enzyme. I was very impressed when reading the papers how processive is this enzyme. A shift from 1.5-2 kb to 4-5 kb does make me think if at least for some RNA-seq libraries this enzyme should be used. I do single-cell RNA-seq but I frequently run RNA-seq on few hundred cells, never more than 500 so it seems almost perfect for those samples. I presume you have done your tests on purified RNA? You are saying that there is no way to make it work on single cells, but for higher than single cell amounts of RNA it might works as long as one strips the enzyme from DNA. It seems that you have just used the same approach as you did for Tn5, otherwise it just doesn't work?
                Did you get to sequence any of those test libraries, I would also like to understand if a 1.5-2 kb and 4-5 kb library will differ substantially after sequencing? Would you recommend this enzyme for library preps in some particular cases or SSII is good enough?

                Thanks a lot, it's always great to talk to you and learn more about RNA-seq.
                Hi,
                exactly, I just tried it on RNA, never on cells (and never sequenced anything, unfortunately). Since the input was still so much higher than what I wanted, I never pursued the experiments further. It might have worked eventually but I didn´t have the time back then (we were finalising the Tn5 paper in those days). I think it is worth trying it anyway with few hundreds cells, it will be very interesting to see what people get when using it! And I believe that, sooner or later, someone will find a way to make it working on single cells too. Hopefully we won´t get stuck with inefficient retroviral RT forever!

                As they say on the website, the enzyme is very sticky and you really need to go through the NaOH-HCL-precipitation/column procedure or the excess of salt will inhibit your PCR. The same approach used for Tn5 (SDS) didn´t really work, if I remember correctly (should go back to my notes, though).
                Good luck! I would be really interesting in knowing how it is working for you, if you are willing to share some information
                /Simone

                Comment


                • Clontech SMARTscribe

                  Originally posted by wishingfly View Post
                  Hi Kneu, since you @me, I will reply here with my two cents with the choice of RTase. Sorry for the delay, I was off from the bench for a vacation.

                  As to the RT efficiency (or if we could say, enzyme activity), in my hand, SuperScript II > ProtoScript II> PrimeScript, while Maxima and SuperScript IV do not work at all. Considering the Invitrogen has not fixed the potential contamination yet, we now mainly use ProtoScript II from NEB.

                  We did have some communication with Invitrogen, and they acknowledged that they have recieved similar complains from other customers in U.S., and now they are investigating the issue. I would encourage all of us in U.S. to contact the customer service in your region and complain your issue, so as to push Invitogen to fix the problem as soon as possible.

                  The Invitrogen also mentioned that the product out of U.S. should be fine, because it comes from a different facility. However, current sales system don't allow for ordering the "non-U.S." version. So we researchers in U.S. have to be patient till they fix the problem.
                  Hello everybody and especially Wishingfly,

                  I followed your advice on complaining to Life Technologies about the SSII contamination. Their European facility also distributes contaminated Superscript II as I found out in my results. Their reply seemed to me a little arrogant (or ignorant) as they said they have had no other complaints regarding this issue ("I’m very sorry to hear that there seems to be a contamination in the recent batches #1688365 and #1688366 and I will look into this in detail. So far, we have received no other complaints about the concerned lots regarding a contamination now any other issues."). They would get back to me as soon as possible... That was written on 07-09.

                  As we are still waiting for a solution, in the meantime we already succesfully switched to ClonTech's SMARTscribe (cat: 639536). Even with 21 Amp cycles the profiles of the negative controls (0 cells) are looking nice and clean. It might help other users as well. Also a big High-Five to Simone for supplying this protocol and all your help and tips, Thanks!
                  Attached Files

                  Comment


                  • Originally posted by JJMS View Post
                    Hello everybody and especially Wishingfly,

                    I followed your advice on complaining to Life Technologies about the SSII contamination. Their European facility also distributes contaminated Superscript II as I found out in my results. Their reply seemed to me a little arrogant (or ignorant) as they said they have had no other complaints regarding this issue ("I’m very sorry to hear that there seems to be a contamination in the recent batches #1688365 and #1688366 and I will look into this in detail. So far, we have received no other complaints about the concerned lots regarding a contamination now any other issues."). They would get back to me as soon as possible... That was written on 07-09.

                    As we are still waiting for a solution, in the meantime we already succesfully switched to ClonTech's SMARTscribe (cat: 639536). Even with 21 Amp cycles the profiles of the negative controls (0 cells) are looking nice and clean. It might help other users as well. Also a big High-Five to Simone for supplying this protocol and all your help and tips, Thanks!
                    I am still convinced that SMARTScribe is repackaged Superscript II since in my hands they perform exactly the same
                    good that it works for you as well!
                    Btw, I also talked to Life Tech and they also said to me that they had no complaints from other customers (that was back in July or August, I think), but since our SSII is fine I didn´t pursue it further. They probably tell the same thing to everybody, clever guys

                    Comment


                    • Originally posted by Simone78 View Post
                      Hi,
                      exactly, I just tried it on RNA, never on cells (and never sequenced anything, unfortunately). Since the input was still so much higher than what I wanted, I never pursued the experiments further. It might have worked eventually but I didn´t have the time back then (we were finalising the Tn5 paper in those days). I think it is worth trying it anyway with few hundreds cells, it will be very interesting to see what people get when using it! And I believe that, sooner or later, someone will find a way to make it working on single cells too. Hopefully we won´t get stuck with inefficient retroviral RT forever!

                      As they say on the website, the enzyme is very sticky and you really need to go through the NaOH-HCL-precipitation/column procedure or the excess of salt will inhibit your PCR. The same approach used for Tn5 (SDS) didn´t really work, if I remember correctly (should go back to my notes, though).
                      Good luck! I would be really interesting in knowing how it is working for you, if you are willing to share some information
                      /Simone
                      Hi Simone,
                      Glad to know that you have already tried the TGIRT on your hand. I wonder if 0.2% SDS can strip the sticky enzyme off the DNA efficiently. And have you tried SDS along with high temperature incubation and also RNase H/ RNase A treatment? From the bioanalyzer, things seem much better than the retroviral RT. Hope to see its application in single-cell RNA-seq.

                      Gary

                      Comment


                      • Originally posted by kobeho24 View Post
                        Hi Simone,
                        Glad to know that you have already tried the TGIRT on your hand. I wonder if 0.2% SDS can strip the sticky enzyme off the DNA efficiently. And have you tried SDS along with high temperature incubation and also RNase H/ RNase A treatment? From the bioanalyzer, things seem much better than the retroviral RT. Hope to see its application in single-cell RNA-seq.

                        Gary
                        Hi Gary,
                        I just tried with 0.2% SDS, in the same way as I am doing after tagmention to strip the Tn5 off the DNA before PCR and it didn´t work. I also tried SDS + NaOH/HCl (skipping columns or precipitation, as they recommend in the protocol) but, as I said, there was probably too much salt at that point and the PCR didn´t work.
                        /Simone

                        Comment


                        • Originally posted by Simone78 View Post
                          I am still convinced that SMARTScribe is repackaged Superscript II since in my hands they perform exactly the same
                          good that it works for you as well!
                          Btw, I also talked to Life Tech and they also said to me that they had no complaints from other customers (that was back in July or August, I think), but since our SSII is fine I didn´t pursue it further. They probably tell the same thing to everybody, clever guys
                          Hi Simone,
                          Yes it sure looks like that. Our profiles with high input and SMARTscribe indeed match ssII nearly to a 100%. But with single cells SMARTscribe deviates a little. I have to leave out Mg because otherwise a smear around 500 bp's becomes visible. But for now, I'm very happy!

                          Comment


                          • Originally posted by JJMS View Post
                            Our profiles with high input and SMARTscribe indeed match ssII nearly to a 100%. But with single cells SMARTscribe deviates a little. I have to leave out Mg because otherwise a smear around 500 bp's becomes visible.
                            SuperScript II's reaction buffer is 50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl2. ClonTech doesn't say what's in the SMARTScribe mix but according to the documentation from their "legacy" single-cell RNA-seq kit, the first-strand reaction buffer is (at 1X) 50 mM Tris-HCl pH 8.3, 75 mM KCl, 6 mM MgCl2. If that's the same thing they provide when you buy SMARTScribe separately (though it might not be, since they also give you 20 mM DTT instead of 100 mM, as I hope you noticed), then of course you have to adjust the magnesium to compensate. Note that the SMARTScribe enzyme is also provided at 100 U/µL vs. SuperScript II's 200 U/µL.

                            Comment


                            • Originally posted by jwfoley View Post
                              SuperScript II's reaction buffer is 50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl2. ClonTech doesn't say what's in the SMARTScribe mix but according to the documentation from their "legacy" single-cell RNA-seq kit, the first-strand reaction buffer is (at 1X) 50 mM Tris-HCl pH 8.3, 75 mM KCl, 6 mM MgCl2. If that's the same thing they provide when you buy SMARTScribe separately (though it might not be, since they also give you 20 mM DTT instead of 100 mM, as I hope you noticed), then of course you have to adjust the magnesium to compensate. Note that the SMARTScribe enzyme is also provided at 100 U/µL vs. SuperScript II's 200 U/µL.
                              that´s true, thanks for the explanation. I should have said "repackaged Superscript...with a twist" (changed few details to justify the new packaging)
                              However, nowadays most or all of the RT protocols around are done with some kind of MMLV-based enzyme and won´t differ so much from each other. My opinion, at least.

                              Comment


                              • Originally posted by Simone78 View Post
                                that´s true, thanks for the explanation. I should have said "repackaged Superscript...with a twist" (changed few details to justify the new packaging)
                                Well, SuperScript II is wild-type MMLV RTase plus point mutations to kill the RNase H domain. In principle different companies could actually be selling different enzymes with different point mutations...

                                Originally posted by Simone78 View Post
                                However, nowadays most or all of the RT protocols around are done with some kind of MMLV-based enzyme and won´t differ so much from each other. My opinion, at least.
                                Even the fancier enzymes like SuperScript III and Maxima are, as far as I can tell, just engineered to be heat-resistant. That's what makes them more processive: you could run SuperScript II at 50 °C and it would probably be just as fast, except the enzyme would degrade too quickly to be very useful. Unfortunately this engineering tends to kill the C-tailing activity for some reason. But if you aren't using template-switching, mere heat resistance is still a legitimate improvement. (For sequencing I might still worry about increased error rates at the higher temperatures.)

                                Comment

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