NEBNext Ultra II Directional RNA Workflow

Deplete ribosomal RNA from total RNA input using either poly(A) mRNA enrichment or ribosomal RNA depletion methods compatible with the NEBNext Ultra II kit.

Incubate RNA at high temperature with divalent cations to fragment it, then hybridize random hexamer primers and perform first strand cDNA synthesis using reverse transcriptase. Actinomycin D is added to prevent unwanted second strand synthesis.

Incorporate uracil residues into the second strand of cDNA during synthesis using the dUTP method, which enables strand-specific library construction.

Clean up the double-stranded DNA, end-repair to create blunt phosphorylated ends, and add a single adenine overhang to the 3' end. Ligate NEBNext or Illumina-compatible adaptors with thymine overhangs.

Treat the library with USER Enzyme (UDG and Endo VIII) to selectively remove the uracil-containing strand and open hairpin loops if using NEBNext adaptors, leaving a single-stranded library with strand-specificity.

Perform either a cleanup step for smaller fragments or size selection for larger fragments to remove contaminants and unwanted byproducts.

Amplify the library using Q5 High-Fidelity DNA Polymerase with the recommended number of cycles based on input amount. This step selects for dual-adapted molecules, increases yield, and incorporates barcodes and P5/P7 sequencing sequences.

Clean up the amplified library to remove reagents and impurities, resulting in a final strand-specific library ready for cluster generation and sequencing.