3. Applications of AIR Bulk Profiling

Depending on the sample type, total RNA, DNA, or isolated immune cells (see DirectCell™ Protocol) may be used as inputs for the DriverMap AIR Assay. No preliminary poly(A) selection or ribosomal/mitochondrial / globin RNA depletion is required for RNA samples:

• For the AIR RNA assay, the table below describes the optimal input amount and range of total RNA purified from different biological samples. As illustrated in Fig. 15, using less than the optimal amount of RNA could reduce reproducibility in detecting medium-abundant clonotypes.

• For bulk tissue, blood, or cell samples with more than ~50,000 cells, purify RNA and DNA using conventional protocols (e.g., as recommended in the DriverMap AIR user manual). For biological samples with a small number of cells—less than ~50,000 cells (e.g., sorted immune cells) or blood and tissue microsamples—we recommend using the immune cells directly as input as described in our DirectCell Protocol. In the case of samples with limited numbers of cells, the AIR-RNA assay is recommended for use with the DirectCell protocol due to that assay’s higher sensitivity when compared to the AIR-DNA assay.

• For the AIR DNA assay, the general rule for quantitating high- and medium-abundant clonotypes is to use at least 5-10 µg of DNA corresponding to approximately 1 million cells. Using a smaller amount of DNA (0.5-2 µg) would reduce the detection of medium-abundant clonotypes, and even lower amounts (0.1-0.5 µg) would also reduce the sensitivity of detecting high-abundant clonotypes (Fig. 15). Considering the reduced sensitivity of the AIR DNA assay due to the low copy number of starting DNA molecules, technical replicates are highly recommended.