Appendix for use with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB #E7760, #E7765) and NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB #E7770, #E7775)
 

6.1. Fragmentation

Note: These recommendations have been optimized using Universal Human Reference Total RNA. Other types of RNA may require different fragmentation times.

Figure 6.1. Modified fragmentation times for longer RNA inserts.



Red Ladder
Blue 150-300 bp, mRNA fragmented for 15 minutes at 94°C
Green 200-500 bp mRNA fragmented for 10 minutes at 94°C
Cyan 400-1,000 bp mRNA fragmented for 5 minutes at 94°C

Modified fragmentation times for longer RNA inserts. Bioanalyzer traces of RNA as shown in an RNA Pico Chip. mRNA isolated from Universal Human Reference RNA and fragmented with First Strand Synthesis Reaction Buffer and Random Primer Mix (2X) at 94°C for 5, 10 or 15 minutes, and purified using 2.2X volume of Agencourt RNAClean XP Beads. For libraries with RNA insert sizes larger than 300 bp, fragment RNA between 5–10 minutes and remember to increase the incubation at 42°C from 15 to 50 minutes during the first strand cDNA synthesis reaction.


6.2. Size Selection of Adaptor Ligated DNA

Note: Size selection should be done after adaptor ligation and USER digestion.

The size selection protocol is based on a starting volume of 96.5 μl. Size selection conditions were optimized with SPRIselect Beads and NEBNext Sample Purification Beads; however, AMPure XP Beads can be used following the same conditions. If using Ampure XP Beads, please allow the beads to warm to room temperature for at least 30 minutes before use.
 

Please adjust recommended bead volumes for each target size according to Table 6.2. The protocol below is for libraries with a 300 bp insert size (420 bp final library size).

Table 6.2: Recommended size selection conditions for libraries with insert sizes larger than 300 bp.

Note: Size selection for < 100 ng total RNA input is not recommended.

 

LIBRARY PARAMETER

APPROXIMATE INSERT SIZE

300 bp

400 bp

450 bp

Approx. Final Library Size

420 bp

520 bp

570 bp

BEAD VOLUME TO BE ADDED (µl)

1st Bead Selection

25

20

15

2nd Bead Selection

10

10

10

Note: Any differences in insert sizes between the Agilent Bioanalyzer and that obtained from paired end sequencing can be attributed to the higher clustering efficiency of smaller sized fragments.

6.2.1. Vortex SPRIselect Beads or NEBNext Sample Purification Beads to resuspend.

6.2.2. Add 25 μl of resuspended beads to the 96.5 μl ligation reaction. Mix well by pipetting up and down at least 10 times.

6.2.3. Incubate for 5 minutes at room temperature.

6.2.4. Place the tube on an appropriate magnetic rack to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic rack. After the solution is clear (about 5 minutes), carefully transfer the supernatant containing your DNA to a new tube (Caution: do not discard the supernatant). Discard the beads that contain the unwanted large fragments.

6.2.5. Add 10 μl resuspended beads to the supernatant, mix well by pipetting up and down at least 10 times and incubate for 5 minutes at room temperature.

6.2.6. Place the tube/plate on an appropriate magnetic rack to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic rack. After the solution is clear (about 5 minutes), carefully remove and discard the supernatant that contains unwanted DNA. Be careful not to disturb the beads that contain the desired DNA targets (Caution: do not discard beads).

6.2.7. Add 200 μl of 80% freshly prepared ethanol to the tube while in the magnetic rack. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.

6.2.8. Repeat Step 6.2.7 once.

6.2.9. Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic rack with the lid open.

Caution: Do not over-dry the beads. This may result in lower recovery of DNA target. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack, they are too dry.

6.2.10. Remove the tube/plate from the magnetic rack. Elute the DNA target from the beads by adding 17 μl of 0.1 X TE (provided) to the beads. Mix well on a vortex mixer or by pipetting up and down ten times. Quickly spin the tube and incubate for 2 minutes at room temperature.

6.2.11. Place the tube on a magnetic rack. After the solution is clear (about 5 minutes), transfer 20 μl to a new PCR tube for amplification.

6.3. PCR Enrichment of Size-selected Libraries

Note: Size-selected libraries require 2 additional PCR cycles due to loss during size selection steps compared to non-size-selected libraries.

Check and verify that the concentration of your oligos is 10 μM on the label.
 

Use Option A for any NEBNext oligos kit where index primers are supplied in tubes. These kits have the forward and reverse primers supplied in separate tubes.

Use Option B for any NEBNext oligos kit where index primers are supplied in a 96-well plate format. These kits have the forward and reverse (i7 and i5) primers combined.

6.3.1. Set up the PCR reaction as described below based on the type of oligos (PCR primers) used.

6.3.1A. Forward and Reverse Primers Separate

COMPONENT

VOLUME PER ONE LIBRARY

Adaptor Ligated DNA (Step 6.2.11)

15 µl

 (blue) NEBNext Ultra II Q5 Master Mix

25 µl

Universal PCR Primer/i5 Primer*,**

5 µl

Index (X) Primer/i7 Primer*, **

5 µl

Total Volume

50 µl



* NEBNext Oligos must be purchased separately from the library prep kit. Refer to the corresponding NEBNext Oligo kit manual for determining valid barcode combinations.

** Use only one i7 primer/ index primer per sample. Use only one i5 primer (or the universal primer for single index kits) per sample.
 
6.3.1B. Forward and Reverse Primers Combined

COMPONENT

VOLUME PER ONE LIBRARY

Adaptor Ligated DNA (Step 6.2.11)

15 µl

 (blue) NEBNext Ultra II Q5 Master Mix

25 µl

Index (X) Primer/i7 Primer Mix*

10 µl

Total Volume

50 µl



* NEBNext Oligos must be purchased separately from the library prep kit. Refer to the corresponding NEBNext Oligo kit manual for determining valid barcode combinations.

** Use only one i7 primer/ index primer per sample. Use only one i5 primer (or the universal primer for single index kits) per sample.

6.3.3. Place the tube in a thermocycler with the heated lid set to 105°C. Perform PCR amplification using the following PCR cycling conditions:

CYCLE STEP

TEMP

TIME

CYCLES

Initial Denaturation

98°C

30 seconds

1

Denaturation

98°C

10 seconds

 

variable*, **

Annealing/Extension

65°C

75 seconds

Final Extension

65°C

5 minutes

1

Hold

4°C

 



* The number of PCR cycles should be adjusted based on RNA input. Size-selected libraries require additional 2 PCR cycles and should be adjusted accordingly. For example if a non-size selected library requires 8 PCR cycles, the size-selected library should be amplified for 10 cycles (8 + 2) after the size selection.

** It is important to limit the number of PCR cycles to avoid overamplification.

If overamplification occurs, a second peak ~ 1,000 bp will appear on the Bioanalyzer trace.


Figure 6.3: Bioanalyzer traces of size selected DNA libraries.



50 ng mRNA was fragmented with First Strand Synthesis Reaction Buffer and Random Primer Mix (2X) at 94°C for 15, 10 or 5 minutes. Libraries were size-selected as described in Table 4.2, then amplified by PCR, and run on Agilent Bioanalyzer DNA 1000 chip.

Fragmentation times and corresponding size selection conditions are shown in the table below.

 

 

Table 6.3:

LIBRARY SAMPLE

FRAGMENTATION TIME

1st BEAD SELECTION

2nd BEAD SELECTION

Red

10 minutes

25 µl

10 µl

Blue

5 minutes

20 µl

10 µl

Green

5 minutes

15 µl

10 µl


For libraries with longer inserts (> 200 bp), remember to increase the incubation at 42°C from 15 to 50 minutes during the First Strand cDNA Synthesis reaction.