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  • #31
    Hi Simone,

    I've been using your Smart-seq2 protocol to construct over 20 libraries in the last couple of months w/ great results (Arabidopsois root tissue; around few dozens to few hundreds cells, starting w/ ~30pg-3ng total RNA). This week it stopped working. The problem is somewhere in one of the reactions b4 Tagmentation. There is almost no trace for the ~2000bps peak in the BioAnalyzer after pre-amplification reaction. I know this is one of the weird things we, biologist experience and there is usually no straight forward answer but maybe you have any suggestion or noticed that one of the materials is highly sensitive (e.g., freeze and thaw of the KAPA enzyme).

    Besides that, less critical issues, what is the % of mapped reads you get and do you get a lot of rRNA reads (I do). Why did you include the VN nts at the end of the poly(dT) primers and did you try primers that do not have these nts.

    Another question is why not using lower Ampure beads ratio after the KAPA reaction same as after the XT reaction to get rid of all oligos that are <200bps.

    BTW, I've tried to add more of the poly(dT) primers and it seems that less rRNA is present in the final reads, but as I said, currently, nothing works.

    Thanks and have a nice weekend,
    Guy

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    • #32
      Originally posted by wacguy View Post
      Hi Simone,

      I've been using your Smart-seq2 protocol to construct over 20 libraries in the last couple of months w/ great results (Arabidopsois root tissue; around few dozens to few hundreds cells, starting w/ ~30pg-3ng total RNA). This week it stopped working. The problem is somewhere in one of the reactions b4 Tagmentation. There is almost no trace for the ~2000bps peak in the BioAnalyzer after pre-amplification reaction. I know this is one of the weird things we, biologist experience and there is usually no straight forward answer but maybe you have any suggestion or noticed that one of the materials is highly sensitive (e.g., freeze and thaw of the KAPA enzyme).
      Hi Guy,
      Difficult to say why it´s not working. KAPA Pol is not that sensitive. In fact, on their website they say you could even leave it at RT over the weekend and it´ll still work (but don´t do it!). The other reagents are not sensitive either. Maybe your RNAse inhibitor has gone bad or you started using a new batch of primers that are not good (problems in the synthesis)? My only suggestion is to replace ALL the reagents and start over, which I know is a bit annoying.

      Originally posted by wacguy View Post
      Besides that, less critical issues, what is the % of mapped reads you get and do you get a lot of rRNA reads (I do). Why did you include the VN nts at the end of the poly(dT) primers and did you try primers that do not have these nts.
      We usually get 60% uniquely mapping reads, 20% multi-mapping and 20% unmappable. We keep only the uniquely mapping of course and, of those, we have 60% reads mapping to exons, 20% intronic and 20% intergenic. rRNA is never an issue, always <5%, usually around 1-2%.
      We include the "VN" to be sure that the oligo dT will anneal at the beginning of the poly-A tail, thus avoiding an unnecessarily long stretch of A that might cause problems to the reverse transcriptase and, later, to the KAPA Pol.


      Originally posted by wacguy View Post
      Another question is why not using lower Ampure beads ratio after the KAPA reaction same as after the XT reaction to get rid of all oligos that are <200bps.
      Initially that was our intention, but we noticed that we were losing also some of the long fragments along with the short. We are now making our own beads and we change the buffer composition according to the size of fragments we want to recover but it´s never perfect anyway. A solution would be to block the primers, of course. I did limited trials on that but I think that is the only thing that might solve the problem.
      Best,
      Simone

      Comment


      • #33
        Thank you Simone!

        I appreciate your reply, changing EVERYTHING, and checking the PCR machine was and is my next plan.

        Best,
        Guy
        Last edited by wacguy; 06-15-2014, 06:11 AM.

        Comment


        • #34
          for TSO, is HPLC purification necessary for this protocol? and could you recommend any reliable vendors in US for locked nucleic acid. We usually order oligos from IDT, but they don't provide service for LNA. Thanks!

          Comment


          • #35
            Exiqon is the only LNA vendor in North America... though I believe they actually get their orders produced by IDT anyway.

            Comment


            • #36
              Hi konglongjidan,

              I used the same companies mentioned in the SMART2-seq article (Biomers & Exiqon). I work at Duke so I guess you would be able to do the same. They were all HPLC purified, I wouldn't change that.

              Good luck,
              Guy

              Comment


              • #37
                thank you guys for an exciting and very informative exchange of ideas and protocols. smartseq2 with LNA seems to work better than 3rG as mentioned in the paper, at least in my hands. just for information, i get my LNA TSO synthesized by eurogentec (in europe) at 40nmol scale, the yield was 95 micrograms and it cost me 30 euros. so it is not expensive at all.

                Comment


                • #38
                  Simone,
                  Really appreciate all your advice on this forum.
                  How much have you played with the concentration of betaine during the RT, or investigated other duplex destabilizers?
                  Many thanks for any feedback.
                  Eli

                  Comment


                  • #39
                    Also, if one were to leave out the betaine, you would predict a lower yield of library, and bias in the data due to selective loss of reads from transcripts with strong secondary structures?

                    Comment


                    • #40
                      I tried 0.5, 1, 1.5 and 2M betaine (all final conc). While the cDNA yield after preampl was higher for 1M when compared to 0.5M, no further improvements were observed when using more than 1M. Additionally, the highest conc of betaine solution you can buy (or make) is 5M, which means that already 1/5 of your reaction volume is already taken when using 1M. Adding >1M becomes impractical.
                      As we show in the Nat. Method paper, betaine alone is not sufficient but somehow it works only when combined with 6 mM or higher MgCl2 (9-12 mM to maximize yield). I tried betaine in PCR as well (or only in the PCR but no RT) but results were not great and I dropped it. We don´t know if leaving out betaine will cause of lower detection of genes with strong secondary structure, but we do know that we have a worse coverage at the 5´-end of the genes, which might be an indirect proof that we do lose highly structured genes.
                      As alternatives to betaine I tried trehalose (the second best), sorbitol (bad), DMSO (bad) and formamide (bad). For experiments with betaine, trehalose and sorbitol on tot RNA please see Suppl Table 1 in NM paper.
                      Best,
                      Simone

                      Comment


                      • #41
                        TSO with UMI

                        Hi Simone,
                        I have been using your Smart2-seq protocol on single mouse neurons. Based on Bioanalyzer and Fluidigm Biomark with 96 primers looks promising to move forward for sequencing.

                        I wonder whether you have used ERCC as a internal controls, if so what concentration works best in your hands for single cell.

                        I curious to know, (since Linnarsson is in your neighbourhood) have you tested your TSO with UMI for smart2-seq. If so, what is your experience....

                        Best,
                        Suguna

                        Comment


                        • #42
                          From the discussions i've had with C1 users, Spike-ins for single cell are unfortunately very cell-type specific - you want to maintain a 3-5% amount of external RNA to cellular RNA, but the amount of cellular RNA (and lysis efficiency) varies between cell types and even between cells. For practical purposes, though, two papers both used a 40,000 dilution from the Ambion product on the C1.

                          If your cells aren't too rare, you can try to calculate an average RNA mass from bulk. If they are, you may have to do a test run or two to get the ratio right.

                          The good news is that the ERCCs are useful once you get them spiked into your cells ; you can compare the technical variability of ERCCs to that of the measured transcripts/genes to separate biological variability from random noise and calibrate to how much mRNA was present in each cell. Another (slightly off-label) use is also determining the efficiency of reverse transcription.

                          Comment


                          • #43
                            Thanks jparson for your reply.

                            I have used C1 with ERCC dilution you have mentioned above.

                            C1 works best for cultured cells in terms of capturing almost 80-90% of the wells. It is cost effective and more cells to analyze.
                            Whenever I load my primary cells on C1, only 40-50% of the cell get captured. so my cost of prep is high.
                            In addition to that you cannot use different experimental conditions in the same chip. Unless you have two many C1 machines.

                            That is the one of the reasons I moved to alternate manual methods.

                            With Smart2seq, I could able to FACS different cell types and experimental conditions on the same day. Since I want to take advantage of ERCC, I want to know what would be the ideal ERCC concentration for single cell sorting conditions..
                            Now I am using 10 Million final dilution per reaction. I wonder whether this would be enough....

                            Based on Simone's bioanalyzer profile I could nt say whether he used ERCC's and I dont know how he determines his RT effieciency in different cells run at the same experiemental conditions or how to determine the technical variation across different samples...

                            Best,
                            Suguna

                            Comment


                            • #44
                              Originally posted by jparsons View Post
                              From the discussions i've had with C1 users, Spike-ins for single cell are unfortunately very cell-type specific - you want to maintain a 3-5% amount of external RNA to cellular RNA, but the amount of cellular RNA (and lysis efficiency) varies between cell types and even between cells. For practical purposes, though, two papers both used a 40,000 dilution from the Ambion product on the C1.
                              Totally agree here. One has to optimize the amount of ERCC case by case.
                              For "big" cells (cells in culture: MEF, HEK293T, C2C12, P19, HeLa, etc) we use 0.1 ul of a 1:40000 dil., which gives a final conc in RT of 1:4 million. However, when working with extremely small cells (T-cells, for example) we are using a 1:40 million dil., which is still too high because we get 5-10% (!) reads from the spike-ins in the end. Maybe 1:100 million is the right conc?
                              Again, as parsons said, there other factors that play a role: lysis efficiency, intracellular variability, cell cycle stage, how "stressed" your cells are, lysis buffer you are using, etc etc

                              Originally posted by SUGU View Post
                              I curious to know, (since Linnarsson is in your neighbourhood) have you tested your TSO with UMI for smart2-seq. If so, what is your experience....
                              as far as I know they haven´t tried it yet but it´s quite some time that I don´t talk to them. I think the idea was to run the Smart-seq2 on the C1 but so far there had been other problems, such as the fact that the PCR protocol couldn´t be changed. And this was key to get a good preampl because KAPA HiFi requires 98 degrees for denaturation and higher annealing T, while Advantage 2 requires lower T both for denaturation and annealing.

                              Best,
                              Simone

                              Comment


                              • #45
                                ERCC spikes for C1 negative controls: bioanalyser traces needed

                                Hi,

                                For those of you using ERCC spikes in your single cell RNAseq on the C1, do you have examples of bioanalyser traces for negative controls? (i.e. capture sites with no cells in). We are trying to see what a negative control should look like as the ERCC spikes will be amplified so should show on the trace regardless.

                                Comment

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