Figure 1. Optimized RACE-PCR-based approach for TCR sequencing from single cells.

(A) Sketch of the basic principle of the novel single-TCR sequencing strategy. Reverse transcription and exonuclease digest were consecutively done on-slide. Complete reactions were transferred to 96 well plates for tailing, anchor PCR and nested PCR round I and II. Alpha and beta chains were pre-amplified together during anchor PCR and in separate reactions for nested PCRs I and II. To change the reaction conditions for the first four steps of the protocol, the volume was increased to create optimal conditions for the subsequent enzyme. For nested PCR round I and II 1 µl of the previous reation volume was transferred to the next step. (B) Different priming strategies (standard oligo-dT/random hexamers, one gene-specific primer and three gene-specific primers per TCR chain) were tested with and without extra exonuclease-I digest of residual RT-primers. Decreasing numbers of human T cells, from 1000 to 1 as indicated served as template. (C)The anchor PCR step is used to prolong the oligo-dG overhang from the tailing step. PCR was tested without reverse primer in “linear” mode and with reverse primer in “exponential” mode of amplification. (D) Temperature switch during reverse transcription was tested. RT at a constant temperature of 51°C (upper row) was compared with a temperature increase form 51°C for 30 min to 70°C for 20 min (bottom row). (E) In seven independent single-cell PCR experiments a total number of 266 samples were processed. Numbers of samples yielding α- and/or β- chain products above the evaluated full-length cut-off size were calculated as a percentage of total samples per experiment. Mean values for all experiments taken together are indicated by horizontal lines.