| CPC C12N 15/1093 (2013.01) [C07K 16/00 (2013.01); C07K 2317/565 (2013.01)] | 20 Claims |
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1. A method for generating a T-cell receptor repertoire (TCRR) from a cell population comprising a T-cell population, the method comprising:
a) isolating mRNA from said cell population comprising a T-cell population;
b) fragmenting said mRNA to obtain a collection of mRNA fragments having a mean fragment length that is less than 600 bp;
c) preparing cDNA from said collection of fragments;
d) ligating at least a first adapter module to said cDNA; wherein said first adapter module ligates to a first end of said cDNA;
e) performing a first round of PCR amplification using a first primer and a second primer, wherein said first primer binds to a first region and said second primer binds to a second region in said first round of PCR amplification, wherein the first region is at least partially in said first adapter and said second region is in the C-region, thereby obtaining a plurality of first amplified products; and
f) performing a second round of PCR amplification on said plurality of first amplified products using a third primer and a fourth primer thereby deriving a plurality of second amplified products, wherein
said third primer binds to a third region and said fourth primer binds to a fourth region in said second round of PCR amplification,
said third region is at least partially in the first adapter and said fourth region is in the C-region,
an average nucleotide distance between the first and the second region across the plurality of first amplified products is greater than an average nucleotide distance between the third region and the fourth region across the plurality of second amplified products, and
the fourth region is located at least partially between the first and second regions.
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