FAQ: Are libraries prepared with the NEBNext® Small RNA Library prep kit for Illumina compatible with paired-end flow cells for cluster generation?

Yes, libraries prepared by this method can be loaded on paired-end flowcells, as our PCR product is compatible with Illumina’s read 2 sequencing primer. Sequencing primer annealing directions and approximate start sites are depicted by the gray arrows in the schematic below.

small rna primer schematic

To enable paired-end sequencing in the Illumina Experiment Manager Software select “TruSeq RNA v2” or “TruSeq Stranded mRNA” or “TruSeq Stranded total RNA” and change the cycle numbers to the desired number of cycles for your libraries.