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  • Hi,
    in principle you could use the protocol for single cell as it is, provided that the input RNA is not too high. I don´t know exactly, maybe up to 1 ng. If you start form several ng you should increase the amount of some reagents, to avoid their depletion during the RT or PCR. I would (as an example) double the amount of TSO, ISPCR and oligodT primers and increase also the conc of dNTPs. Again, you should run some kind of titration to see what reagent becomes the limiting factor in your reaction. If planning to sort hundreds of cells in each well, you might also consider using a higher conc of Triton to ensure proper lysis (or a stronger buffer like guadine thiocyanate/hydrochloride, RLT, etc). Concentration of Triton of 1-2% do NOT have a negative effect on the RT reaction. If you need extra info just give me more details about your experiments

    Comment


    • Hi all,

      I'm new to single cell RNAseq and just started to worked on SMARTseq2,
      I followed the nature protocol (PMID: 24385147), but one tricky thing is that we don't have enough room to get a individual hood for single cell experiments, so I have to use shared hood with others for RNA operation. For each sample, 10 colorectal cancer cell HT29 cells were picked up with micro manipulator then released into a PCR tube with lysis buffer in tube. After that samples were incubated at 75C for 5min, then followed the SMARTseq2 protocol described in paper, 10 cycles in transcription and 17 cycles in preamplification, but the transcriptase I used was Maxima H Minus Reverse Transcriptase (200 U/µL) (EP0751, Thermo).
      Actually, I got some results that were not too bad at the beginning, but in current few weeks I suffered from severe RNA degradation issue, although the cDNA yield could reach ~80ng, the peak looked really wired, even I doubled the RNase inhibitor, there's no improvement. Is it too harsh to do SMARTseq with my conditions? Hope you experts could help me and give me some suggestions. Thank you!
      Attached is the electropherogram from analyzer https://1drv.ms/u/s!AmIgzq2vDK6fliMiOkXyIEPnfozZ
      Last edited by Yin; 05-16-2019, 04:47 PM.

      Comment


      • I am trying to do single cell RNAseq for human T cells using Smartseq2. I used the smartscribe to do RT and KAPA HiFi to do pre-amplification. I followed the protocol from my colleague, which leaves out 1M Betaine and 6mM MgCl2 for the first 5 samples. Later I heard from another colleague that adding Betaine will increase the cDNA yield so I added these two reagents for the rest of my samples. Other settings and reagents were kept the same. When I did the QC for the samples by fragment analyzer after bead clean-up, I found with Betaine and MgCl2 the average concentration of the preamplified cDNA decreased dramatically, from 0.5ng/uL to 0.03 ng/uL. So I am wondering what could be the problem.

        Comment


        • Originally posted by lijing2019 View Post
          I am trying to do single cell RNAseq for human T cells using Smartseq2. I used the smartscribe to do RT and KAPA HiFi to do pre-amplification. I followed the protocol from my colleague, which leaves out 1M Betaine and 6mM MgCl2 for the first 5 samples. Later I heard from another colleague that adding Betaine will increase the cDNA yield so I added these two reagents for the rest of my samples. Other settings and reagents were kept the same. When I did the QC for the samples by fragment analyzer after bead clean-up, I found with Betaine and MgCl2 the average concentration of the preamplified cDNA decreased dramatically, from 0.5ng/uL to 0.03 ng/uL. So I am wondering what could be the problem.
          That´s really strange, it is generally the opposite: betaine and MgCl2 gives better yield, even when only just one of the two is included. Could you post the Bioanalyzer plots to get an idea of how the cDNA looks like? Although for you I don´t think it´s the case, it is good practice to include a 10 pg total RNA sample as a sort of control, in order to avoid the cell-to-cell variability.
          You are talking about a small number of samples, how did you pick them? By hand? How long the picking take might also have an impact on the RNA quality.
          Best,
          Simone

          Comment


          • Hi Simone,
            I have 4 patients without adding Betaine and MgCl2, the final yields of preamplified cDNA is almost the same, about 0.5ng/uLx25uL. 6 patients with Betaine and MgCl2, one of the patient seemed to have RNA degradation (could you check whether it is RNA degradation?), the other patients all have low concentration, about 0.03ng/uLx25uL. I attached the representative fragment analysis results. Each pateint has 400 cells and I QC 100 cells from each of them, the results are consistent for each protocol.
            I usually thawed the cells and rested them overnight, then FACS sorted them into lysis buffer. I run 22 cycles at the preamplification step. The picking takes about 3 minutes for each plate.
            I am going to try sequence some of them anyway. Do you think the patient which seemed to have RNA degradation will work?
            Last edited by lijing2019; 05-23-2019, 10:07 AM.

            Comment


            • Originally posted by Simone78 View Post
              That´s really strange, it is generally the opposite: betaine and MgCl2 gives better yield, even when only just one of the two is included. Could you post the Bioanalyzer plots to get an idea of how the cDNA looks like? Although for you I don´t think it´s the case, it is good practice to include a 10 pg total RNA sample as a sort of control, in order to avoid the cell-to-cell variability.
              You are talking about a small number of samples, how did you pick them? By hand? How long the picking take might also have an impact on the RNA quality.
              Best,
              Simone
              Hi Simone,
              I have 4 patients without adding Betaine and MgCl2, the final yields of preamplified cDNA were almost the same, about 0.5ng/uLx25uL. 6 patients with Betaine and MgCl2, one of the patient seemed to have RNA degradation (could you check whether it is RNA degradation?), the other 5 patients all have low concentration, about 0.03ng/uLx25uL. I attached the representative fragment analysis results, ERCC were added in every well. Each pateint has 400 cells and I QC 100 cells from each of them, the results are consistent for each protocol.
              I usually thawed the cells and rested them overnight, then FACS sorted them into lysis buffer. I run 22 cycles at the preamplification step. The picking takes about 3 minutes for each plate.
              I am going to try sequence some of them anyway. Do you think the patient which seemed to have RNA degradation will work?
              Attached Files

              Comment


              • Originally posted by lijing2019 View Post
                Hi Simone,
                I have 4 patients without adding Betaine and MgCl2, the final yields of preamplified cDNA were almost the same, about 0.5ng/uLx25uL. 6 patients with Betaine and MgCl2, one of the patient seemed to have RNA degradation (could you check whether it is RNA degradation?), the other 5 patients all have low concentration, about 0.03ng/uLx25uL. I attached the representative fragment analysis results, ERCC were added in every well. Each pateint has 400 cells and I QC 100 cells from each of them, the results are consistent for each protocol.
                I usually thawed the cells and rested them overnight, then FACS sorted them into lysis buffer. I run 22 cycles at the preamplification step. The picking takes about 3 minutes for each plate.
                I am going to try sequence some of them anyway. Do you think the patient which seemed to have RNA degradation will work?
                From what I can see you seem to do everything correctly. You might get something out of the degraded cDNA (yes, it seems degraded to me) but you will also have a very strong 3´bias. The Tn5 transposase will tagment anything <40 bp, cDNA as well as primer dimers.
                Best,
                Simone

                Comment


                • Hello Simone and all,
                  Thank you for the deep thought and informative discussion. I learned a lot. I have a question regarding the oligos for the reactions. It looks like the consensus now is to order biotin blocked oligos. Can I still use standard desalt or I have to order HPLC purified ones?

                  Comment


                  • Originally posted by zebrafish_juju View Post
                    Hello Simone and all,
                    Thank you for the deep thought and informative discussion. I learned a lot. I have a question regarding the oligos for the reactions. It looks like the consensus now is to order biotin blocked oligos. Can I still use standard desalt or I have to order HPLC purified ones?
                    I personally prefer HPLC-purified oligos when working with single cells but desalted would work as well.
                    Best,
                    Simone

                    Comment


                    • RNA-Seq

                      RNA-Seq, also called RNA sequencing, is a particular technology-based sequencing technique which uses next-generation sequencing to reveal the presence and quantity of RNA in a biological sample at a given moment, analyzing the continuously changing cellular transcriptome.

                      Comment


                      • Smart Seq2 Lymphocyte Advice

                        Hi Simone & All,

                        I appreciate this thread has been quiet for a while but I hope someone out there is still occasionally reading it.

                        I have recently been attempting to carry out Smart Seq2 with single memory B cells. I can very efficiently clone out Ig genes via PCR following the pre-amplification suggesting I have at least some good quality cDNA but my traces appear dominated by primer dimer. I have been following the protocol exactly as published in 2012 Nature protocols and wish I had found this thread sooner as it has plenty of great tips especially for adapting the protocol to work with lymphocytes and avoiding the particular issue I am having.

                        In terms of obtaining good quality cDNA with minimal primer dimer following pre-amplification and clean-up it looks like the advice has changed over the years and having gone through every post I have decided to make the following adaptions for working with resting memory B cells which I suspect are going to have very little starting RNA to work with and I just wanted to check that I have understood all of these adaptions correctly?

                        1) Add 5’ Biotin to TSO, Oligo-dT30VN, and ISPCR primers

                        2) Titrate the number of pre-amp cycles, typically around 23-24 cycles for lymphocytes but will vary depending on the cell

                        3) Try titrating the Oligo-dT primer but do not alter the concentration of the TSO or ISPCR primers

                        4) Using an oligodT with "V" and not "VN" in the end

                        5) Make your own magnetic beads (according to the Rohland & Reich, Genome Res 2010 paper) and using a buffer with a lower % of PEG.
                        a. I was wondering if anyone knew how much of a difference this made? I’m sure it is more straightforward than it sounds but I like the ease of just going straight to the AMPure beads, but happy to make all alterations that will help my results


                        Any help anyone can offer would be much appreciated!

                        Best wishes,

                        Luke

                        P.S Thank you all for your wonderful input over the years to this thread, it has been so useful and filled a lot of gaps in our troubleshooting where we have sadly lacked replies from scientists in our local area working with this technique.
                        Last edited by Luke017; 03-04-2020, 01:53 AM.

                        Comment


                        • Originally posted by Luke017 View Post
                          Hi Simone & All,

                          I appreciate this thread has been quiet for a while but I hope someone out there is still occasionally reading it.

                          I have recently been attempting to carry out Smart Seq2 with single memory B cells. I can very efficiently clone out Ig genes via PCR following the pre-amplification suggesting I have at least some good quality cDNA but my traces appear dominated by primer dimer. I have been following the protocol exactly as published in 2012 Nature protocols and wish I had found this thread sooner as it has plenty of great tips especially for adapting the protocol to work with lymphocytes and avoiding the particular issue I am having.

                          In terms of obtaining good quality cDNA with minimal primer dimer following pre-amplification and clean-up it looks like the advice has changed over the years and having gone through every post I have decided to make the following adaptions for working with resting memory B cells which I suspect are going to have very little starting RNA to work with and I just wanted to check that I have understood all of these adaptions correctly?

                          1) Add 5’ Biotin to TSO, Oligo-dT30VN, and ISPCR primers

                          2) Titrate the number of pre-amp cycles, typically around 23-24 cycles for lymphocytes but will vary depending on the cell

                          3) Try titrating the Oligo-dT primer but do not alter the concentration of the TSO or ISPCR primers

                          4) Using an oligodT with "V" and not "VN" in the end

                          5) Make your own magnetic beads (according to the Rohland & Reich, Genome Res 2010 paper) and using a buffer with a lower % of PEG.
                          a. I was wondering if anyone knew how much of a difference this made? I’m sure it is more straightforward than it sounds but I like the ease of just going straight to the AMPure beads, but happy to make all alterations that will help my results


                          Any help anyone can offer would be much appreciated!

                          Best wishes,

                          Luke

                          P.S Thank you all for your wonderful input over the years to this thread, it has been so useful and filled a lot of gaps in our troubleshooting where we have sadly lacked replies from scientists in our local area working with this technique.
                          Hi,
                          actually someone is still reading this thread
                          To answer your questions:
                          1- that's good!
                          2- 23-24 cycles is perfect, we never did more than that.
                          3- that might help as well. I would even increase the TSO conc and see if things get better, maybe from 1 uM to 2 uM.
                          4- debatable, don't agree with that but many people have a different opinion
                          5- lower PEG = lower recovery of small fragments, but also loss of longer fragments if the % is reduced too much. Something in the range 17-21% PEG 8K should be fine. You can also play the DNA:bead ratio, in case.

                          Please take a look at the updated Smart-seq2 in Meth.Mol.Biol., in case you haven't seen it: https://www.ncbi.nlm.nih.gov/pubmed/31028630

                          Good luck!
                          Simone

                          Comment


                          • Perfect, thank you so much for the advice, I really appreciate it and will put it all into action!

                            Best wishes,

                            Luke

                            Comment


                            • PhiX Spike Recommendations

                              Hi All,

                              I just wanted to say thank you for the help, with all of the tips in this thread I have now managed to generate good qualit cDNA and what should hopefully be some nice libraries!

                              I was wondering if anyone could help me out with one final question though, I have been working solely with memory B cells, and I have sent the libraries to our genomics facility. The genomics facility got in touch to say that they use 1% PhiX spike in for their sequencing runs but that low diversity libraries normally require a bit more. I think my final library is going to be pretty low diversity so I was wondering if anyone had any recommendations on what PhiX % to try?

                              Any help would again be much appreciated!

                              Best wishes,

                              Luke

                              Comment


                              • The random tagmentation (or sonication/ligation) will give it plenty of diversity. Lower diversity library types needing extra PhiX are things like amplicons, microbiome 16S, and WGBS.

                                Comment

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