NEB Next Ultra RNAseq Questions: Guide to Library Prep, Barcoding, and RNA Handling Techniques

RNAseq (NEB Next Ultra) Questions: A Detailed Guide

RNAseq using the NEB Next Ultra kit involves multiple critical steps, including RNA preparation, library construction, and sequencing setup—each influencing the data quality. The main variability arises from RNA and library preparation. Using individual barcodes for each replicate is essential for multiplexing. Purification beads such as AMPure or SPRI perform similarly in cleanup. RNA storage conditions vary by tissue type and do not always require RNase inhibitors or rapid freezing. Proper mRNA enrichment or rRNA removal prevents contamination. Handling tissue consistently is fundamental to obtaining reliable RNA sequencing results.

Variability in RNA and Library Preparation

RNAseq data quality can fluctuate substantially based on how the RNA is isolated and how the library is prepared. According to experts, the largest source of variation comes from these early steps rather than sequencing itself. To minimize variability, pooling samples for RNA isolation in a single batch is recommended.

• Freeze biological samples immediately after collection.
• Process all samples together during RNA extraction.
• Perform library preparation steps in parallel when possible.

This approach reduces batch effects and technical variation, leading to more reproducible results.

Barcodes and Oligo Tags Usage in Multiplexing

Multiplexing different samples in one sequencing run requires unique oligonucleotide barcodes, also called tags.

Each biological replicate and experimental condition needs its own barcode:

Sample Group	Number of Replicates	Number of Barcodes Needed
Control	3	3
Experimental	3	3
Total	6

Assign one unique barcode per replicate per condition. This setup allows differentiation of samples after sequencing and avoids mixing reads.

Selection and Use of Purification Beads

Purification during RNAseq library prep is crucial to clean up fragments after adapter ligation and to perform size selection. Various bead types such as AMPure, SPRI, and NEBNext purification beads are commonly used.

• All these beads function comparably for cleaning and size selection.
• Agilent AMPure beads are noted for effective size selection and sample cleanup.
• Using beads after adapter ligation improves library quality by removing unligated fragments.

The choice of beads mostly depends on availability and cost because performance is similar.

Detailed Protocol Steps for NEB Next Ultra RNAseq Library Prep

One validated procedure integrates mRNA fragmentation, cDNA synthesis, and adapter ligation with purification:

1. Fragmentation: Use RNAse III to fragment mRNA into suitable sizes.
2. First strand synthesis: Use random hexamers and M-MuLV Reverse Transcriptase.
3. Second strand synthesis: Apply NEBNext second strand synthesis module.
4. End repair and adapter ligation: Prepare ends and ligate sequencing adapters.
5. Size selection: Perform cleanup and size selection with AMPure beads.

This workflow is adaptable for platforms like Ion Torrent and Illumina.

RNA Storage: RNase Inhibitors and Temperature Preferences

RNA integrity depends on storage conditions and tissue type:

• Tissues with high ribonuclease activity might require special treatments during isolation.
• RNase inhibitors are generally not necessary in the storage buffer if the isolation is clean.
• Storage buffers can be simple—water, TE buffer, or sodium citrate work well.
• Freezing samples at -80°C preserves RNA quality for long-term storage.

Flash freezing in liquid nitrogen is optional and does not significantly impact final RNA quality. RNA can be safely stored frozen without RNA stabilization reagents like RNAlater if handled properly.

Flash Freezing vs RNAlater Usage

Choosing between flash freezing and RNAlater depends on convenience and tissue type:

• Flash freezing with liquid nitrogen allows prompt tissue preservation and immediate grinding if desired.
• Storing tissue at -80°C after flash freezing is adequate for most sample types.
• RNAlater can preserve RNA in samples awaiting processing, particularly for delicate tissues prone to degradation.
• If extraction happens immediately, RNAlater may not provide additional benefits.
• Consistent sample handling between controls and treated samples maintains experimental reliability.

Fresh vs Frozen Tissue Handling for RNAseq

The decision to use fresh or frozen tissue affects workflow flexibility rather than RNA quality:

• Effective homogenization is essential regardless of tissue state.
• Freezing samples before RNA extraction generally does not degrade RNA quality significantly.
• Treat tissues consistently per lab protocols to minimize processing variability.

Empirical testing has shown minimal differences in RNA quality between fresh and frozen tissues if handled properly.

Strategies for mRNA Enrichment and Ribosomal RNA Removal

Enriching for mRNA or depleting ribosomal RNA is a critical cost and quality control step in library preparation:

• Poly(A) selection with magnetic beads (e.g., Thermo Fisher’s MagJET mRNA Enrichment Kit) effectively enriches samples for mRNA.
• Ribosomal RNA removal ensures sequencing reads target coding regions, avoiding wasted sequencing effort on abundant rRNA.
• Failure to remove rRNA leads to large proportions of reads mapping to ribosomal sequences, reducing usable data.
• The choice between poly(T) selection and rRNA depletion depends on sample type and experimental goals.

Summary of Key Considerations for NEB Next Ultra RNAseq Experiments

• Prepare all RNA samples simultaneously to reduce batch effects and variability.
• Use unique barcodes per replicate and condition to enable multiplexed sequencing.
• Agilent AMPure beads provide effective cleanup post-adapter ligation.
• RNA storage at -80°C without inhibitors is generally sufficient if isolation is clean.
• Flash freezing tissue aids preservation but is not mandatory for all sample types.
• Consistent treatment between control and experimental samples is key to reliable comparison.
• mRNA enrichment or rRNA depletion steps significantly improve sequencing efficiency.
• Focus on proper tissue handling and homogenization for best RNA quality.

Addressing these factors enhances the reliability and reproducibility of RNA sequencing results using the NEB Next Ultra kits.

Q1: How many barcodes do I need for my RNAseq samples using NEB Next Ultra?

You need one barcode per replicate or sample. For example, 2 conditions with 3 replicates each require 6 barcodes total.

Q2: Are AMPure beads different from SPRI or NEBNext beads for purification?

They are essentially the same. AMPure beads are effective for size selection and cleaning after adapter ligation.

Q3: Is flash freezing with liquid nitrogen necessary for RNA storage?

Flash freezing is optional. Storing samples at -80°C without liquid nitrogen often produces good RNA quality.

Q4: Do I need RNase inhibitors during RNA storage?

Usually not required. As long as the isolation process is done properly, storing RNA in standard buffers without inhibitors works well.

Q5: How do I reduce ribosomal RNA contamination in my RNAseq library prep?

Use mRNA enrichment kits like Thermo Fisher’s polyT beads. Without rRNA removal, most reads may map to ribosomal RNA.