5.1. General DNA Preparation Protocol

Follow the guidelines below to isolate and quantify DNA for larger bulk tissue, blood, or cell samples. For comprehensive AIR repertoire analysis, including identification of antigen-activated clonotypes and expression profiling, consider purifying both RNA and DNA from the same biological samples.

Use the appropriate method to isolate genomic DNA. We recommend the following DNA isolation kits.

• Whole blood: we recommend using the Tempus (Thermo-Fisher) or PAXgene (QIAGEN) for DNA isolation. Based on our experience, the collection of 3 ml of whole blood in Tempus RNA Blood Tubes followed by RNA isolation using Tempus Spin RNA Isolation kit and DNA from the supernatant using ethanol precipitation after the first centrifugation step provides the most simple and reliable protocol for purification of both RNA and DNA from whole blood (Ferrante J.A, et al. A novel technique for isolating DNA from Tempus blood RNA tubes after RNA isolation. BMS Res, Notes (2018) 11:53).

• PBMC, buffy coat, or fresh/frozen tissue: for more than 50,000 cells, we recommend using the QIAGEN All Prep DNA/RNA micro/mini kit for both DNA and RNA purification, DNeasy Blood & Tissue kit for DNA purification and RNAeasy Micro/Mini Plus Kit for total RNA isolation. For tissue samples with low content of immune cells, please consider purifying lymphocyte cell fraction before DNA isolation to increase the sensitivity of AIR DNA repertoire profiling.

• FFPE tissue: we recommend using the Qiagen All Prep DNA/RNA FFPE kit. With FFPE the yield and quality of isolated DNA/RNA can vary significantly, depending on the age and method used to fix the sample.