5.3. DirectCell™ Protocol

As shown in the Table above, for a small number of cells, such as immune cell fractions isolated by FACS or magnetic beads (e.g. 5,000-50,000 cells), and tissue samples (up to ~1 mg), we recommend using cells directly without RNA purification step. Using sorted cells/tissues increases the sensitivity of CDR clonotype detection as compared to RNA-isolated samples. Refer to the guidelines in this section for the preparation of these samples.

To use small samples directly in the assay, follow the procedure below:

1. Purify immune cells by FACS sorting—
   Collect cells in 1xPBS or any commonly used buffer without magnesium. Transfer the cells into 1.5-ml or 0.5-ml test tubes, spin down at low speed (e.g., 500g for 5 min), and remove excess supernatant from the cell pellet, keeping the residual volume in the test tube 14 ul (to prevent cell loss due to complete removal of supernatant).
   OR
   Prepare dissociated tissue samples (e.g., by collagenase treatment). Adjust the volume of each sample to 14 ul. Depending on your cell collection method, this may require spinning down the cells and removing excess supernatant over 14 µl.

2. When you are ready to start the DriverMap AIR Assay, prepare the Hybridization Buffer Master Mix as described in Step 1 of the Hybridization Procedure, add 1 µl of 2% N-Lauroyl Sarcosine, and then set up the Hybridization reaction with your sample as described.

3. Follow the standard protocol for hybridization, AMP purification, cDNA synthesis, and amplification steps.