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I am wondering if anyone who is well versed with VDJ sequencing for TCR repertoire analysis (specifically CDR3) would know if DNA or RNA is a better starting material? We are looking at the effects of radiation therapy on immune system of mice. Thanks

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  • $\begingroup$ DNA is typically easier to work with. Just make sure the cells you're working with have actually matured enough and undergone VDJ recombination. $\endgroup$ – MattDMo Jan 29 at 2:14
  • $\begingroup$ I agree with @MattDMo Colleagues next door have done this with single sorted cells which where sequenced afterwards. $\endgroup$ – Chris Jan 29 at 7:15
  • $\begingroup$ @MattDMo @Chris♦ how do you suggest one makes sure T cells have matured and undergone VDJ recombination?? I have looked at literature and seen one study that looked at clonal expansion 22 days after radiation exposure but they did not say why. Also, I have read that RNA is better to use than DNA since DNA won’t give you the antigen specificity you can see with mRNA expression. I have also seen that DNA can only show you the possible combinations of VDJ you could get as mRNA shows you the ones you did get, in real time. $\endgroup$ – Manon Valiquette Jan 29 at 18:12
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    $\begingroup$ I suggest you spend some time looking at the details of somatic recombination. Early during maturation, the V, (D,) J, and C protogenes recombine in the DNA to form the first version of the recombined TCR $\alpha/\gamma$ and $\beta/\delta$ chains (different protogenes combine in different ways in the different chains). These recombined sequences are then transcribed as mRNA - to my knowledge the protogenes are never transcribed. So, mRNA sequences should match DNA sequences. $\endgroup$ – MattDMo Jan 30 at 22:59
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    $\begingroup$ The reason I gave my caveat is because if you just take a whole thymus and do single-cell sequencing, you'll also get immature T cells that haven't undergone recombination. Instead, I would use either blood-derived cells, or sort thymocytes using a marker that is expressed after recombination. I would choose blood, because some of the recombined thymocytes will die during further maturation, and probably shouldn't be included in your measurements of TCR diversity. $\endgroup$ – MattDMo Jan 30 at 23:01

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