TIRTL-seq 384-well v2

Résumé fourni par la source

TIRTL-seq is a method to obtain paired TCR-sequencing information from millions of human T cells. It is based on splitting cells into 96-384 wells, preparing and sequencing TCRalpha and TCRbeta libraries from each well in miniaturized reactions, and then pairing alpha and beta chains based on co-occurrence patterns or relative frequency variations between wells. For more details about the method, please see our paper (https://www.nature.com/articles/s41592-025-02907-9). The 384-well protocol requires some automation: a non-contact liquid dispenser (we have tested Dispendix I.Dot mini/S/L and Formulatrix Mantis) and a device to transfer 1 uL between 384-well plates (we use Integra Viaflo, but many other solutions should work equally well). We have also designed a 96-well protocol with increased volumes, which does not require any automation but has lower resolution. Schematic of TIRTL-seq protocol. Briefly, a cell suspension is distributed into 384-well plates containing RT/lysis mastermix under a hydrophobic overlay using non-contact liquid dispensers. After the RT reaction, PCR I mastermix with V-segment and C-segment primers is dispensed into the same plate. The PCR I product is then diluted and transferred to the PCR II plate for indexing PCR with well-specific unique dual indices. The PCR II products are pooled by centrifugation, purified, size-selected using magnetic beads, and sequenced on an Illumina platform. Total library preparation cost is listed for one 384-well plate.