text: Each candidate CRM (cCRM) is cloned upstream of a reporter gene. Compared to traditional reporter assays, the main innovation is the use of fluorescence activated cell sorting (FACS) of dissociated cells, instead of microscopy, to screen for tissue-specific enhancers. This approach utilizes a two-marker system: in each embryo, one marker (here, the rat CD2 cell surface protein) is used to label cells of a specific tissue for being sorted by FACS, and the other marker (here, green fluorescent protein GFP) is used as a reporter of CRM activity.
  Cells are sorted according to their tissue type and then by GFP fluorescence, and the cCRMs are recovered by PCR from double-positive sorted cells, and from total input cells. High-throughput sequencing of both populations then allows measuring the relative abundance of each cCRM in input and sorted populations; one can then assess the enrichment or depletion of each cCRM in double-positive cells versus input as a measure of activity in the CD2-positive cell type being tested.