Nebnext ultra 2 rna nondirectional library prep kit for

Paramagnetic beads coupled with oligo d(T) are combined with total RNA to isolate poly(A) + transcripts based on NEBNext^® Poly(A) mRNA Magnetic Isolation Module manual. Prior to first strand synthesis, samples were randomly primed (5′ d(N6) 3′ [N = A,C,G,T]) and fragmented based on manufacturer’s recommendations (NEBNext^® Ultra™ II RNA Nondirectional Library Prep Kit for Illumina^®). The first strand is synthesized with the Protoscript II Reverse Transcriptase with a longer extension period (40 min for 42 °C). All remaining steps for library construction were used according to the NEBNext^® Ultra™ II RNA Nondirectional Library Prep Kit for Illumina^®. Illumina 8-nt dual-indices were used. Samples were pooled and sequenced on a HiSeq with a read length configuration of 150 PE.

Paramagnetic beads coupled with oligo d(T) are combined with total RNA to isolate poly(A)+ transcripts based on NEBNext® Poly(A) mRNA Magnetic Isolation Module manual. Prior to first strand synthesis, samples were randomly primed (5′ d(N6) 3′ [N=A,C,G,T]) and fragmented based on the manufacturer’s recommendations (NEBNext® Ultra™ II RNA Nondirectional Library Prep Kit for Illumina®). The first strand is synthesized with the Protoscript II Reverse Transcriptase with a longer extension period (40 min for 42 °C). All remaining steps for library construction were used according to the NEBNext® Ultra™ II RNA Nondirectional Library Prep Kit for Illumina®. Illumina 8-nt dual-indices were used. Samples were pooled and sequenced on a HiSeq with a read length configuration of 150 PE.