The NEBNext High-Input Poly(A) mRNA Magnetic Isolation Module is designed to isolate intact poly(A)+ RNA from high inputs (5-50 µg per 0.2 ml reaction) of previously isolated total RNA, using oligo d(T)25-coupled paramagnetic beads. Intact mRNA can be obtained in approximately one hour, and eluted in small volumes.
The NEBNext High-Input Poly(A) mRNA Magnetic Isolation Module isolates intact poly(A)+ RNA from high inputs (5-50 µg per 0.2 ml reaction) of previously isolated total RNA. The technology is based on the coupling of oligo d(T)25 to paramagnetic beads, which is then used as the solid support for the direct binding of poly(A)+ RNA. The procedure permits the manual processing of multiple samples and can be adapted for automated high-throughput applications. Additionally, magnetic separation technology permits elution of intact mRNA in small volumes, thereby eliminating the need for precipitation of the poly(A)+ transcripts in the eluent. Intact poly(A)+ RNA, which is representative of the mRNA population of the original sample, can be obtained in approximately one hour.
For inputs less than 5 µg, the NEBNext® Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) is recommended.
Figure 1: The NEBNext High Input Poly(A) mRNA Isolation Module provides consistent representation across a wide input range
A. Poly(A) RNA enriched using the NEBNext High Input Poly(A) mRNA Isolation module from 50 μg or 5 μg of total Universal Human Reference (UHR) RNA (Agilent®) containing External RNA Controls Consortium (ERCC) RNA and Spike-In RNA Variants (SIRV Set 4, Lexogen®). Total yield was assessed using Qubit® RNA High Sensitivity Assay Kit (Invitrogen®). Poly(A) RNA is typically ~1-5% of total RNA.
B. Bioanalyzer® (Agilent) traces for Total UHR RNA and poly(A)-enriched RNA from 50 μg or 5 μg total RNA input.
C. Transcript abundances were determined from libraries prepared from 40 ng (50 μg input) or 8 ng (5 μg input) poly(A)-enriched RNA using the NEBNext Ultra™ II Directional RNA Library Prep Kit and sequenced on an Illumina® NextSeq® 550 sequencer. Six million reads were sampled from each library.
Figure 2: The NEBNext High Input Poly(A) mRNA Isolation Module produces low ribosomal RNA retention, across sample types
Poly(A) RNA was enriched using Dynabeads® mRNA Purification Kit (Invitrogen), Poly(A)Purist™ MAG (Invitrogen) or the NEBNext High Input Poly(A) mRNA Magnetic Isolation Module from 50 μg Universal Human Reference RNA (UHR, Agilent) or RNA extracted from mouse kidney tissue or S. cerevisiae (yeast) using the Monarch® Total RNA Miniprep Kit.
A. Representative Bioanalyzer traces of total and poly(A)-enriched RNA.
B. Percent ribosomal RNA (rRNA) of total or poly(A)-enriched RNA samples was determined from sequencing of triplicate (UHR and mouse poly(A) samples) or duplicate (total RNA and yeast poly(A) RNA samples) experiments, with standard deviation. Libraries were prepared from 40 ng poly(A)-enriched RNA using the NEBNext Ultra™ II Directional RNA Library Prep Kit and sequenced on an Illumina NextSeq 550 instrument. Six million reads were sampled from each library.
Figure 3: RNA from the NEBNext High Input Poly(A) mRNA Isolation Module produces higher library yields for nanopore sequencing, with good read mapping
400 ng of poly(A)-enriched Universal Human Reference (UHR) RNA, enriched using the stated methods, was prepared for Direct RNA Sequencing (ONT #SQK-RNA002) on a GridION® sequencer (Oxford Nanopore Technologies®). 100 ng of poly(A)-enriched UHR RNA, enriched using the stated methods, was prepared for Direct cDNA Sequencing (ONT #SQK-DCS109) on a GridION sequencer.
A. Library yields were assessed using Qubit dsDNA High Sensitivity Assay Kit (Invitrogen); shown are the average of replicates with standard deviation.
B. Average mapping percentages of reads from replicate Direct RNA and Direct cDNA sequencing runs with standard deviation.
Figure 4: The NEBNext High-Input Poly(A) mRNA Isolation Module yields high-quality poly(A) mRNA for direct RNA and cDNA Sequencing
5´➝3´ transcript coverage of the top one thousand transcripts from representative Direct RNA (A) and Direct cDNA (B) sequencing runs on the Oxford Nanopore Technologies platform with libraries prepared from poly(A)-enriched UHR RNA using the NEBNext High Input Poly(A) mRNA Isolation Module.
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