TITLE: E7805 NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina® Protocol for use with Inputs ≤ 100 ng
AUTHORS: jbonnevie

[DESCRIPTION]
The NEBNext Ultra II FS DNA Module contains the enzymes and buffers required to convert a broad range of input amounts of DNA into high quality libraries for next generation sequencing on the Illumina platform. The fast, user friendly workflow also has minimal hands-on time. 
Note: The Ultra II FS Kit is not compatible with bisulfite conversion workflows. 
Each kit component must pass rigorous control standards, and for each new lot the entire set of reagents is functionally validated together by construction and sequencing of indexed libraries on an Illumina sequencing platform.
For larger volume requirements, customized and bulk packaging is available by purchasing through the OEM/Bulks department at NEB. Please contact OEM@neb.com for further information.


[GUIDELINES]
Safe Stop: This is a point where you can safely stop the protocol and store the sample prior to proceeding to the next step.

Caution: Signifies a step in the protocol that has multiple paths leading to the same end point but is dependent on a user variable, like the amount of input DNA. 

Color: Indicates the cap color of the reagent to be added to a reaction.

The NEBNext Ultra II FS DNA Module is Designed for use with the Following:
NEBNext Ultra II Ligation Module (NEB #E7595)
NEBNext Ultra II Q5® Master Mix (NEB #M0544)
NEBNext Singleplex or Multiplex Oligos for Illumina® www.neb.com/oligos
This protocol is written for non-UMI adaptors


[STEPS]
SECTION: Starting Material
1. 100 pg–500 ng purified, genomic DNA. We recommend that the DNA be in 1X TE (10 mM Tris pH 8.0, 1 mM EDTA), however, 10 mM Tris pH 7.5–8, low EDTA TE or H2O are also acceptable. If the input DNA is less than 26 µl, add TE (provided) to a final volume of 26 µl. 

SECTION: Fragmentation/End Prep
2. Fragmentation occurs during the 37 °C incubation step.
 
Use the chart below to determine the incubation time required to generate the desired fragment sizes. Incubation time may need to be optimized for individual samples. See Figure 1.1 for a typical fragmentation pattern. 
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SECTION: Fragmentation/End Prep
3. Ensure that the Ultra II FS Reaction Buffer is completely thawed. If a precipitate is seen in the buffer, pipette up and down several times to break it up, and quickly vortex to mix. Place on ice until use.
  
 


SECTION: Fragmentation/End Prep
4. Vortex the Ultra II FS Enzyme Mix 5-8 seconds prior to use and place on ice. 
  
 

Figure 1.1: Example of size distribution on a Bioanalyzer®. Human DNA (NA19240) was fragmented for 5-40 min.
 




SECTION: Fragmentation/End Prep
5. Add the following components to a 0.2 ml thin wall PCR tube on ice:


 
  
  

SECTION: Fragmentation/End Prep
7. In a thermal cycler, with the heated lid set to 75 °C, run the following program. 
 
 

Safe Stop: If necessary, samples can be stored at -20 °C; however, a slight loss in yield (~20%) may be observed. We recommend continuing with adaptor ligation before stopping.

SECTION: Adaptor Ligation
10. Set a 100 μl or 200 μl pipette to 50 µL and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube.
 
(Caution: The NEBNext Ultra II Ligation Master Mix is very viscous. Care should be taken to ensure adequate mixing of the ligation reaction, as incomplete mixing will result in reduced ligation efficiency. The presence of a small amount of bubbles will not interfere with performance.)
SECTION: Adaptor Ligation
11. Incubate at 20 °C for 15 min in a thermal cycler with the heated lid off.

SECTION: Adaptor Ligation
12. Add 3 µL of (red or blue) USER® Enzyme to the ligation mixture from the previous step.
  
 


SECTION: Adaptor Ligation
13. Mix well and incubate at 37 °C for 15 min with the heated lid set to ≥ 47 °C.

 
SECTION: Size Selection or Cleanup of Adaptor-ligated DNA
14. Caution: The following section is for cleanup of the ligation reaction for inputs ≤ 100 ng. If your input DNA is > 100 ng, follow the size selection protocol in Chapter 2, Section 2.3. If you want fragment sizes > 550 bp and your input is ≥ 100 ng, follow the entire protocol in Chapter 3. 
 



SECTION: Size Selection or Cleanup of Adaptor-ligated DNA
15. Vortex SPRIselect or NEBNext Sample Purification Beads to resuspend.  

SECTION: Size Selection or Cleanup of Adaptor-ligated DNA
16. Add 57 µL (0.8X) resuspended beads to the Adaptor Ligation reaction. Mix well by pipetting up and down at least 10 times. Be careful to expel all of the liquid out of the tip during the last mix. Vortexing for 3-5 seconds on high can also be used. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.

SECTION: Size Selection or Cleanup of Adaptor-ligated DNA
17. Incubate samples at room temperature for at least 5 min.

SECTION: Size Selection or Cleanup of Adaptor-ligated DNA
18. Place the tube/plate on an appropriate magnetic stand 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 stand.
SECTION: Size Selection or Cleanup of Adaptor-ligated DNA
19. After 5 min (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets
 
(Caution: do not discard the beads). 
SECTION: Size Selection or Cleanup of Adaptor-ligated DNA
20. Add 200 µL of 80% freshly prepared ethanol to the tube/plate while in the magnetic stand. Incubate at room temperature for 30 s, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
SECTION: Size Selection or Cleanup of Adaptor-ligated DNA
21. Repeat the previous step once for a total of two washes. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.
SECTION: Size Selection or Cleanup of Adaptor-ligated DNA
22. Air dry the beads for up to 5 min while the tube/plate is on the magnetic stand with the lid open.
 

SECTION: Size Selection or Cleanup of Adaptor-ligated DNA
23. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 17 µL 0.1X TE (dilute 1X TE Buffer 1:10 in water).
  

SECTION: Size Selection or Cleanup of Adaptor-ligated DNA
24. Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 min at room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.

SECTION: Size Selection or Cleanup of Adaptor-ligated DNA
25. Place the tube/plate on the magnetic stand. After 5 min (or when the solution is clear), transfer 15 µL to a new PCR tube.

SECTION: Size Selection or Cleanup of Adaptor-ligated DNA
26. Proceed to PCR Enrichment of Adaptor-ligated DNA in Next Section.
 
Safe Stop: Samples can be stored at -20 °C.

SECTION: PCR Enrichment of Adaptor-ligated DNA
27. 

Select PCR Reaction Setup
SECTION: PCR Enrichment of Adaptor-ligated DNA
28. Set a 100 μl or 200 μl pipette to 40 µL and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube.

SECTION: PCR Enrichment of Adaptor-ligated DNA
29. Place the tube on a thermal cycler with the heated lid set to 105 °C and perform PCR amplification using the following PCR cycling conditions:


 


 


 


SECTION: PCR Enrichment of Adaptor-ligated DNA
30. Proceed to Cleanup of PRC reaction in next section.
SECTION: Cleanup of PCR Reaction
31.  
SECTION: Cleanup of PCR Reaction
32. Vortex SPRIselect or NEBNext Sample Purification Beads to resuspend.
SECTION: Cleanup of PCR Reaction
33. Add 45 µL (0.9X) resuspended beads to the PCR reaction. Mix well by pipetting up and down at least 10 times. Be careful to expel all of the liquid out of the tip during the last mix. Vortexing for 3-5 seconds on high can also be used. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.

SECTION: Cleanup of PCR Reaction
34. Incubate samples on bench top for at least 5 min at room temperature.

SECTION: Cleanup of PCR Reaction
35. Place the tube/plate on an appropriate magnetic stand 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 stand.

SECTION: Cleanup of PCR Reaction
36. After 5 min (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets
 
Caution: do not discard the beads.
SECTION: Cleanup of PCR Reaction
37. Add 200 µL of 80% freshly prepared ethanol to the tube/plate while in the magnetic stand. Incubate at room temperature for 30 s, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
SECTION: Cleanup of PCR Reaction
38. Repeat the previous step once for a total of two washes. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.
SECTION: Cleanup of PCR Reaction
39. Air dry the beads for up to 5 min while the tube/plate is on the magnetic stand with the lid open.
 


SECTION: Cleanup of PCR Reaction
40. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 33 µL of 0.1X TE (dilute 1X TE Buffer 1:10 in water).

SECTION: Cleanup of PCR Reaction
41. Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 min at room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.

SECTION: Cleanup of PCR Reaction
42. Place the tube/plate on the magnetic stand. After 5 min (or when the solution is clear), transfer 30 µL to a new PCR tube and store at -20 °C.

SECTION: Assess Library Quality on a Bioanalyzer
43. Dilute library (from the previous step) 5-fold in 0.1X TE Buffer (inputs ≤ 1 ng may not require dilution to run on a Bioanalyzer).

SECTION: Assess Library Quality on a Bioanalyzer
44. Run 1 µL on a DNA High Sensitivity Chip.

SECTION: Assess Library Quality on a Bioanalyzer
45. Check that the library size shows a narrow distribution with an expected peak size based on fragmentation time (Figure 1.2).
 
Figure 1.2: Example of final library size distribution without size selection. Human DNA (NA 19240) was fragmented for 5-40 min. 
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SECTION: Adaptor Ligation
9. Add the following components directly to the FS Reaction Mixture 


 
* Mix the Ultra II Ligation Master Mix by pipetting up and down several times prior to adding to the reaction.
** The NEBNext adaptor is provided in the NEBNext Oligo Kits. NEB has several oligo options which are supplied separately from the library prep kit. Please see www.neb.com/oligos for additional information.
  
  
  

 

SECTION: Adaptor Ligation
8. Determine whether adaptor dilution is necessary.
 
Caution: If DNA input is < 100 ng, dilute the (red) NEBNext Adaptor for Illumina in 10 mM Tris-HCl, pH 7.5-8.0 with 10 mM NaCl as indicated in Table 1.2.1. 
 
Table 1.2.1: Adaptor Dilution

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SECTION: Fragmentation/End Prep
6. Vortex the reaction for 5 s and briefly spin in a microcentrifuge.