RNAseq (Illumina MiSeq or NextSeq 2000, Pacbio Iso-seq)

The number of studies involving transcriptomics continues to increase. The mRNA can be enriched in order to make RNA sequencing more cost effective. In eukaryotes (and any other species with polyadenylated mRNA) the mRNA is enriched using the NEBNext Poly(A) mRNA Isolation Module. Good quality RNA is needed for this protocol (RIN >7).

For bacteria (or any other species without poly-adenylated mRNA) an rRNA depletion kit  will be used to remove the rRNA, leaving all other RNA in the library. There are different kits that can be used on eukaryote RNA as well to deplete the rRNA, if one is interested in other RNA, not just the mature mRNA.

The mRNA enriched or rRNA depleted RNA is then used to make the RNAseq libraries. The kit we presently use is the NEBNext® Ultra™ II Directional RNA Library kit. For precious or limited amounts of RNA, the NEBNext SingleCell/Low Input RNALibrary Prep Kit is also available.

With PacBio Iso-seq, it is possible, with good quality RNA, to sequence the entire transcript which makes isomers easier to detect. However, throughput is lower than the Illumina platforms.