Protocol for Non-directional RNA-seq Workflows and NEBNext® Ultra™ II RNA First Strand Synthesis Module (E7771)

This is a point where you can safely stop the protocol and store the samples prior to proceeding to the next step in the protocol.
This caution sign signifies a step in the protocol that has two paths leading to the same end point but is dependent on a user variable, like the type of RNA input. 
Colored bullets indicate the cap color of the reagent to be added

RNA Sample Recommendations
RNA Integrity:
RNA Integrity Number (RIN) is computed using ribosomal RNA (rRNA) amount in the sample. If rRNA is removed by any method, the RIN value should not be used to evaluate the integrity of the RNA sample. In this case, we recommend that the fragmentation time is empirically determined if the RNA sample is suspected to be low quality. The following recommendation apply to the total RNA samples only.

Assess the quality of the input RNA by running the RNA sample on an Agilent Bioanalyzer RNA 6000 Nano/Pico Chip to determine the RNA Integrity Number (RIN). RNA with different RIN values require different fragmentation times or no fragmentation at all.

For intact (RIN > 7) or partially degraded RNA samples (RIN = 2 to 7) follow the First Strand Synthesis Protocol in Chapter 2 (current chapter). See Table 2.1.1 for the recommended fragmentation times.

For highly degraded samples (RIN = 1 to 2) (e.g. FFPE), which do not require fragmentation, follow the library preparation protocol in the NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB #E7770) manual.

RNA Purity:
The RNA sample should be free of DNA, salts (e.g., Mg2+, or guanidinium salts), divalent cation chelating agents (e.g. EDTA, EGTA, citrate), or organics (e.g., phenol and ethanol).

Input Amount Requirement
1 ng – 100 ng total RNA purified mRNA or rRNA depleted RNA that is quantified after the purification. RNA should be DNA free in up to 5 µl of Nuclease-free Water, quantified by Qubit Fluorometer and quality checked by Bioanalyzer.

The protocol is optimized for approximately 200 nt RNA inserts. To generate libraries with longer RNA insert sizes, refer to Appendix A (Chapter 6) for recommended fragmentation times and size selection conditions.

This protocol has been optimized using Universal Human Reference Total RNA.

Note: This protocol is for total RNA, purified mRNA or rRNA depleted RNA only. For use with the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) or the NEBNext rRNA Depletion Kit (NEB #E6310) and the NEBNext Ultra II RNA Library Prep Workflow, please follow the protocol in the NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB #E7770) manual.

2.1. RNA Fragmentation and Priming

RNA fragmentation is only required for intact or partially degraded RNA. Recommended fragmentation times can be found in Table 2.1.1.

2.1.1. Assemble the fragmentation and priming reaction on ice in a nuclease-free tube by adding the following components:

 Purified mRNA or rRNA Depleted RNA  5 µl
(lilac) NEBNext First Strand Synthesis Reaction Buffer
 4 µl
(lilac) Random Primers  1 µl
 Total Volume  10 µl

2.1.2. Mix thoroughly by pipetting up and down several times.

2.1.3. Place the sample in a thermal cycler and incubate the sample at 94°C following the recommendations in Table 2.5.1 below for fragment sizes ~200 nt.

Table 2.1.1 Suggested fragmentation times based on RIN value of RNA input.
RNA Type RIN Frag. Time Intact RNA > 7 15 min. at 94°C Partially Degraded RNA 2–6 7–8 min. at 94°C

Intact RNA > 7 15 min. at 94°C
Partially Degraded RNA 2-6 7–8 min. at 94°C

Note: Refer to Appendix A for fragmentation conditions if you are preparing libraries with large inserts (> 200 bp). Conditions in Appendix A (Chapter 3) only apply for intact RNA.

2.1.4. Immediately transfer the tube to ice and proceed to First Strand cDNA Synthesis.

2.2 First Strand cDNA Synthesis Reaction

2.2.1. Assemble the first strand synthesis reaction on ice by adding the following components to the fragmented and primed RNA from Step 2.1.4:

 Fragmented and primed RNA (Step 2.1.4)  10 µl
 Nuclease-free Water  8 µl
(lilac) NEBNext First Strand Synthesis Enzyme Mix  2 µl
 Total Volume  20 µl

2.2.2. Mix thoroughly by pipetting up and down several times.

2.2.3. Incubate the sample in a preheated thermal cycler with the heated lid set at ≥ 80°C as follows:

Note: If you are following recommendations in Appendix A (Chapter 6), for longer RNA fragments, increase the incubation at 42°C from 15 minutes to 50 minutes at Step 2.

Step 1: 10 minutes at 25°C
Step 2: 15 minutes at 42°C
Step 3: 15 minutes at 70°C
Step 4: Hold at 4°C

2.2.4. Proceed directly to Second Strand cDNA Synthesis, using the NEBNext Ultra II Non-directional RNA Second Strand Synthesis Module (NEB #E6111).