GSM8074617: MBC2, replicate 1, spatial TCR sequencing; Homo sapiens; OTHER
To capture spatial TCR clonotype information, we adapted an established protocol that allows spatial mapping of TCRs from cDNA libraries of our samples. The process involves three qPCR steps:  1. First step begins with pooled 43 TCRB primers and truncated Read 1 primer (2uL of cDNA, 1 uL of each forward and reverse primers, and 12.5 uL of NEBNextMasterMix, 0.5uL Sybr, and 8ul water). The PCR is stopped before the plateau phase and cleaned with 0.8X Amupure beads.  2. Second step uses 43 TCRB primers with R2 sequences and truncated Read 1 primer with 1uL of PCR product from step 1.  3. Third step involves indexed Truseq P5 primers and indexed Nextera P7 primers, with 1uL of PCR product from step 2.  Sequencing was conducted on an Illumina NextSeq 500 with the following cycle settings: R1 28, I1 10, I2 10, R2 110. Clonotype analyses were performed with MiXCR. The PCR cycling conditions are as follows:  -Initial Denaturation: 98°C for 3 minutes -Denaturation: 98°C for 15 seconds -Annealing: 62°C (72°C for qPCR step 3) for 20 seconds -Extension: 72°C for 1 minute -Final Extension: 72°C for 1 minute