5.2. Instructions for Spike-in Controls

• Before using AIR Spike-In Controls Mixes or Isoform Pools, heat the whole test tube(s) containing controls at 70°C for 3 minutes, cool down to room temperature, and pipet the whole content of the test tube(s) by pipet several (5-10) times. The heating/mixing step is important to achieve uniformity in the concentration of Spike-In Controls aliquots.

• Directly add an aliquot of Spike-In Control Mix to the RNA sample before performing any processing step using AIR-RNA or similar AIR-seq assays. We recommend using a positive control RNA sample in every experiment to help troubleshoot, analyze data, and normalize across different sample runs. To achieve high reproducibility, avoid adding volumes of less than 1 µl. For RNA samples with low-content immune cells, it may be necessary to dilute Spike-In Controls with the dilution buffer provided in the kit.

*Heating and pipetting are recommended after each freezing cycle to generate homogeneous RNA control concentration.