The NEXTFLEX® Combo-Seq™ Kit enables the user to generate combined mRNA and microRNA libraries from 5 ng – 100 ng of total RNA, without the need for rRNA depletion or poly(A) selection. The workflow relies on a novel technique in which poly(A)-tailed RNA species are selectively reverse transcribed then sheared by RNase H into fragments of useful length. The mRNA fragments are subsequently processed along with the small RNAs present. Libraries prepared with this kit are directional (strand-specific) and compatible with Illumina® sequencers. This kit utilizes a 5’ adapter with a randomized 3’ end to greatly reduce sequence bias in library construction, allowing more accurate identification and quantification of miRNAs, piRNAs, snoRNAs and other small RNAs. The 5’ adapter also features a novel inverted and dideoxy-terminated “anchor” region that further reduces ligation bias. Dilution of the 5' adapter is not required, even at inputs as low as 5 ng of total RNA. NEXTFLEX® Combo-Seq™ sequencing libraries contain Unique Dual Indices (or UDIs), which are designed to specifically address the index-hopping phenomenon associated with Illumina® platforms utilizing a patterned flow cell. These UDIs prevent mis-assigned reads from appearing in final datasets, allowing for the highest assurance of data integrity.
The NEXTFLEX® Combo-Seq™ Kit contains enough material to prepare 48 RNA samples for Illumina® next-generation sequencing platforms. The shelf life of all reagents is 6 months when stored properly. All components can safely be stored at -20°C, except: NEXTFLEX® Adapter Depletion Solution, Resuspension Buffer, and Nuclease-free Water, which can be stored at room temperature, and NEXTFLEX® Cleanup Beads, which should be stored at 4°C.
Learn more about the NEXTFLEX® Combo-Seq™ Kit