I am wondering if someone has experience they can share (also privately if preferable) about different kits/methods to sequence the T-cell repertoire from RNA derived from non-lymphoid tissue (e.g. lung, colon). Specifically, if anyone has had experience with Adaptive, iRepertoire, clonotech or others I'd be happy to discuss.
Thank you!
Hi Asaf,
Are you looking for ways to isolate your T cells from tissues, or just in the repertoire analysis one?
For isolating T cells from non-lymphoid tissue, there should be extensive info. Most likely involving collagenase-based tissue disruption. I'd suggest checking Donna Farber's papers.
For analysis of the repertoire we used to do these in my old lab:
Article T Cell Receptor Diversity Inversely Correlates with Pathogen...
This method is quite robust and really not hard to set-up. You'd need a bunch of primers, a machine that can do single-cell sort (most can these days) and a PCR machine. The beauty of this method is that you get paired ab-TCR by and it just uses sanger seq.
Similarly, you can use the same approach for gdTCRs, it just uses different primers:
Article Rapid cloning, expression, and functional characterization o...
I don't have experience with commercial kits for TCR-repertoire analysis. As it happens, now I have to do mouse BCR-repertoire analysis! I was recommended a commercial kit from TAKARA, based on deep-seq. They also have it for looking into huTCRs.
https://www.takarabio.com/products/next-generation-sequencing/immune-profiling/human-tcr-profiling-kit-for-illumina-sequencing
Best wishes :)
Full disclosure: I'm biased against all of these companies because I work in a lab where we generate our own repertoire data, and I'm often asked by outside people to analyze the crappy data these companies produce. I honestly cannot recommend any of the companies that you mentioned. What I find particularly problematic about their approaches is that they refuse to release all of the data that they generate for you; they cut out large and important parts of the sequence in order to obscure the primers they use for targeted sequencing. Anyway, what you want to do depends entirely on what you're starting with. If you have viable tissue and you want paired TCR (e.g., paired alpha-beta sequences on a per-cell level), I recommend that you team up with someone with access to a 10x Chromium box for single-cell VDJ sequencing. If you don't care about having paired repertoire information, you can very cheaply do bulk TCR repertoire with UMIs using using widely available protocols. We tend to use this protocol https://www.nature.com/protocolexchange/protocols/5011#/procedure with some alterations we've made here and there for further optimization. Feel free to get in touch if you want to discuss further.
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