RNA-mediated oligonucleotide annealing, selection, and ligation with next-generation sequencing (RASL-Seq) is a 2-dimensional RNA sequencing method to quantify expression profiles of several hundred genes, under thousands of different conditions.
Custom probe pairs are designed for each gene of interest. A pair of probes needs to contain: 1) One probe containing a common index primer on its 3' end and a 20 nt oligonucleotide corresponding to the targeted exon sequence with a phosphate on the 5' end; and 2) another probe with a P5 adapter on its 5' end with a 20 nt sequence complementary to the exon that is adjacent to the other probe. The probe pairs are hybridized to the mRNA and separated from total RNA using oligo(dT)-biotin beads. A ligation step joins the probe pairs into a single PCR amplicon probe. The biotinylated mRNA strands are subsequently attached to streptavidin magnetic beads to elute the probe fragments. Next, P7 adapters are attached to the 3' index primer, and the library undergoes PCR amplification before sequencing. The library is sequenced from the 40 nt ligated P5 primer, followed by sequencing from the P7 primer oligonucleotide.