5.1. General Guidelines

AIR Repertoire Profiling:

• AIR TCR-BCR profiling Kit provides reagents for unbiased, comprehensive profiling of all seven immune receptor chains in one reaction and could be used for a wide range of biological samples containing both T and B cells. AIR TCR-BCR kit is a good starting assay for samples with unknown lymphocyte compositions. For samples that contain only T or B cells (e.g., purified immune cell fractions), we recommend using individual TCR or BCR profiling kits.

Starting Amount of RNA:

The table below provides a general guideline for starting amount of RNA for AIR TCR/BCR profiling assay:

• The optimal range for AIR assay is 25-100 ng of total RNA from a large number of immune cells e.g., PBMC, buffy coat, whole blood, purified immune cell fractions (more than 50,000), or lymphoid tissues with a high percentage of immune cells. For PBMC RNA we recommend using 50 ng, and for whole blood RNA, we recommend 100 ng.

• Although the standard AIR protocol works reliably in the 1 ng-1,000 ng range, for deep, quantitative AIR repertoire profiling, we recommend using 25-100 ng of total RNA (from whole blood, PBMC samples, or isolated immune cells). Using larger amounts of total RNA can improve the detection of rare clonotypes, but it requires increasing the sequencing depth (and NGS cost) which can affect expression levels of high-medium abundant clonotypes. Using smaller amounts of total RNA will reduce the number of clonotypes that can be reliably profiled. For example, running 5-10 ng of total whole blood/PBMC RNA instead of 50 ng will profile ~200-500 highly abundant clonotypes (i.e., transcripts with >10 target CDR mRNA molecules) as opposed to ~1,000 clonotypes.

• For RNA isolated from tissue samples with a low percentage of immune cells (e.g., cancer biopsies, non-lymphoid tissues), the amount should be increased to use 200-1,000 ng of total RNA. Due to the high specificity of the AIR assay, the total RNA can be used directly without purification of immune cell fraction.

Replicates:

• For samples with enough total RNA, we recommend running replicates or increasing the amount of total RNA at the hybridization step (e.g., 3-fold) and splitting the samples after the Forward Primer extension step, and running the first and second amplification steps in triplicate reactions for each sample. This allows estimation of variability and expands confident quantitation from ~500 clonotypes if a single replicate is used to ~1,000-3,000 clonotypes (in PBMC) based on the statistical power gained with triplicate analyses (see also the Guide to AIR profiling for more details).

• For purified immune cell samples, we do not recommend splitting the samples into triplicates since the number of cells is usually limited. We recommend collecting multiple samples for each cell fraction so the biological replicates can be run in parallel.

PBMC RNA is supplied with the kit as a positive control. We recommend running at least one positive control sample with a similar concentration to your experimental samples. This will help with troubleshooting, data analysis, and normalization across different sample runs.

When running DriverMap AIR and T/B500 Immune Marker assays on the same immune cell sample (e.g. in sorted T/B cell fractions), we suggest running them separately by splitting the sample by approximately 5:1 ratio as this will ensure the highest sensitivity for each assay. At NGS step, amplified AIR and T/B marker fractions could be mixed (at different ratios, e.g., 1:1 molar ratio) to provide balanced, high sensitivity detection of both AIR CDR and T/B marker gene expression levels.