5.2. DirectCell™ Protocol

For a small number of cells such as immune cell fractions or a small number of cells grown in 96-well plates (e.g. 500-10,000 cells), and tissue samples (up to ~1 mg), we recommend directly using cells in the DriverMap EXP Assay without an RNA purification step. 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 cell fractions (e.g. by FACS) or prepare dissociated tissue samples (e.g., by collagenase treatment). Adjust the volume of each sample to a total of 12 µl. Depending on your cell collection method, this may require spinning down the cells and removing excess supernatant over 13 µl. Any type of cell collection buffer (e.g., 1x PBS or cell growth media) is compatible with the DirectCell protocol.
   

2. When you are ready to start the assay, prepare the Hybridization Buffer Master Mix as described in Step 1 of the Hybridization Procedure but add NP-40 to 0.1% to facilitate cell lysis, and then set up the Hybridization reaction with your sample as described.

3. Follow the standard protocol for hybridization, SPRIselect magnetic bead purification of RNA-Rev GSP hybrids, cDNA synthesis, and amplification steps.