Figure 1. RainDrop BS-seq workflow. RainDrop BS-seq allows targeted, high-throughput bisulfite sequencing using the microdroplet-based PCR amplification system developed by RainDance Technologies. The custom primer panel for the genomic regions of choice is prepared by RainDance Technologies. The workflow comprises the following key steps: (1) bisulfite conversion of genomic templates; (2) merger of picoliter-volume droplets of bisulfite-treated templates with pre-made primer pair droplets (primer panel) on microfluidic chips; (3) pooled, thermal cycling of the PCR reactions (microdroplet PCR); (4) destabilization of droplets to release the PCR products; (5) purification of PCR products using magnetic beads; (6) incorporation of DNA sequencing barcodes through standard PCR (universal PCR), followed by purification of the PCR products and Illumina sequencing. The workflow comprises a two-step tailed primer strategy. First, target sequences (A) are amplified with tailed primers containing a partial Illumina adapter sequence (B). Second, the remainder of the Illumina adapter sequence as well as a unique barcode sequence (C) is added to enable multiplexing of samples for next-generation DNA sequencing.