Input DNA Requirements
Starting material is purified DNA with the following characteristics:
• Input amount 100 pg to 500 ng.
• Insert size 25 bp to 500 bp.
• If you want to perform enzymatic fragmentation of DNA, we recommend using the NEBNext Ultra II FS DNA Module (#E7810). This kit include reagents for fragmentation, end repair, and dA-tailing (see manufacturer's instructions). Note that if performing enzymatic fragmentation, starting volumes of input DNA in the library prep protocol are slightly different, and some optimization is necessary.
• Input DNA should be repaired after fragmentation and have a dA tail. If your fragmented DNA (mechanical or enzymatic) is not end-repaired and dA-tailed, we recommend using the NEBNext Ultra II End Repair/dA-Tailing Module (# E7546). See manufacturer's instructions on how to perform end repair and dA-tailing.
• DNA dissolved in one of these buffers:
— 1×IDTE Buffer (10 mM Tris pH 8.0, 0.1 mM EDTA)
— TE (10 mM Tris pH 8.0, 1 mM EDTA or lower)
— Molecular-grade nuclease-free water.
Ligate Adapter
Ligate the Singular Genomics SG Universal Library Prep Adapter to the fragmented, dA-tailed DNA.
1. For input DNA < 100 ng: Dilute the adapter to 1.5 µM or 0.6 µM with nuclease-free water, depending on the amount of input DNA at the fragmentation step:
Reagent
Less than 5 ng input DNA
5–99.9 ng input DNA
100–500 ng input DNA
SG Universal Library Prep Adapter (15 µM)
1 µL
1 µL
Use adapter undiluted
Nuclease-free water
24 µL
9 µL
Total
25 µL
10 µL
Final concentration
0.6 µM
1.5 µM
2. Pipette the NEBNext Ultra II FS Ligation Master Mix up and down 10 times before use to mix.
The presence of a small number of bubbles will not interfere with performance.
3. Add adapter, NEBNext Ultra II FS Ligation Enhancer, and NEBNext Ultra II Ligation Master Mix to the tube with fragmented DNA.
Reagent
Volume
Fragmented DNA
35 µL
NEBNext Ultra II Ligation Master Mix
30 µL
NEBNext Ligation Enhancer
1 µL
SG Universal Library Prep Adapter
2.5 µL
Total
68.5 µL
Note
If you are setting up multiple reactions, you can first prepare a master mix with NEBNext Ultra II Ligation Master Mix, NEBNext Ligation Enhancer, and SG Universal Library Prep Adapter. Multiply the volumes of these reagents by the number of reactions and make sure to add 10% to the volumes to account for pipetting inaccuracies. Then add 33.5 µL of the mix to the 35 µL of fragmented DNA.
4. Pipette up and down 10 times to mix thoroughly and spin down briefly.
5. Incubate for 15 minutes at 20° C on a covered block or thermocycler with the heated lid off.
Cleave Loop
Next, the loop of the adapter is cleaved by excising the cleavable site in the adapter loop with the Cleave Enzyme as provided in the kit from Singular Genomics.
6. Add cleave reagents to the tube with adapter-ligated DNA.
Reagent
Volume
Adapter-Ligated DNA
68.5 µL
Nuclease-Free Water
3.5 µL
4× Cleave Reaction Buffer
25 µL
Cleave Enzyme
3 µL
Total
100 µL
Note
If you are setting up multiple reactions, you can first prepare a master mix on ice with nuclease-free water, 4× Cleave Reaction Buffer, and Cleave Enzyme. Multiply the volumes of these reagents by the number of reactions and make sure to add 10% to the volumes to account for pipetting inaccuracies. Then add 31.5 µL of the mix to the 68.5 µL of adapter-ligated DNA.
7. Mix thoroughly by pipetting up and down 10 times and spin down briefly.
8. Set the heated lid of a thermocycler to 105° C and run the following program:
Temperature
Time
Incubate
37° C
10 min
Incubate
67° C
30 min
Hold
4° C
Hold
After the samples have cooled, proceed to the cleanup step.
Cleanup after Adapter Ligation
After the adapter ligation and cleaving the loop ends, you need to clean up the sample with magnetic beads. The cleanup can be done using SparQ, SPRI, or Ampure magnetic beads, or another magnetic beads purification kit.
Note
The guidelines are a starting point and you may need to optimize purification ratio depending on the size of your library. For more information, see the Size Selection section in the G4™ Best Practices and Quality Control Guide.
9. Equilibrate magnetic beads to room temperature for 15-30 minutes prior to use.
10. Vortex magnetic beads for 30 seconds.
11. Add 0.8× ratio of magnetic beads to each tube with adapter-ligated sample:
Reagent
Volume
Sample
100 µL
Magnetic beads
80 µL
TOTAL
180 µL
12. Mix well by pipetting up and down 10 times.
13. Incubate at room temperature for 5 minutes.
14. Place samples on magnetic rack, wait until solution is clear, approximately 1–2 minutes.
15. Carefully aspirate and discard the supernatant using a P200 pipette without disturbing the beads.
16. Add 200 µL freshly prepared 80% ethanol to the beads on the magnet.
18. Repeat step 15–17 for a total of 2 washes.
19. Remove supernatant using a P200 pipette, then remove all remaining ethanol with a P10 or P20 pipette without disturbing the beads. Let beads dry for two minutes.
Note
Do not let beads over-dry.
20. Take tubes off magnet and add 17 µL of TE to each tube.
21. Mix well by pipetting up and down 10 times.
22. Incubate samples for 5 minutes.
23. Return to magnet and wait until solution is clear.
24. Transfer 15 µL of supernatant to new PCR tube without disturbing the beads.
You can store the samples at -20° C but Singular Genomics recommends proceeding to PCR amplification.
Singular Genomics Products
You need to have Singular Genomics reagents with SG Universal Library Prep Adapter and cleave reagents. Order one of the following:
Part Numbers
Consumable
Supplier
Purpose
700,110
SG Universal Library Prep Adapter + UDI Primers [1–96]
Adapter, primers, 4X Cleave Reaction Buffer, Cleave Enzyme
700,111
SG Universal Library Prep Adapter + UDI Primers Set A [1–24]
Adapter, primers, 4X Cleave Reaction Buffer, Cleave Enzyme
700,112
SG Universal Library Prep Adapter + UDI Primers Set B [25–48]
Adapter, primers, 4X Cleave Reaction Buffer, Cleave Enzyme
700,118
SG Universal Library Prep Adapter (24 Rxns)
Adapter, 4X Cleave Reaction Buffer, Cleave Enzyme (700,118)
700,117
SG Universal Library Prep Adapter (96 Rxns)
Adapter, 4X Cleave Reaction Buffer, Cleave Enzyme (700,117)
Laboratory Consumables
Consumable
Supplier
Purpose
Ethanol 200 proof (absolute) for molecular biology
General lab supplier
Washing beads
Molecular-grade nuclease-free water
General lab supplier
Various dilutions
One of these magnetic bead cleanup kits:
SparQ PureMag
SPRIselect
AMPure XP
Reaction cleanup and size selection
0.2 mL thin-walled PCR tube
General lab supplier
Reactions, cleanup, dilutions.
Disposable gloves, powder-free
General lab supplier
General purpose