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.

17. Incubate for 1 minute.

18. Repeat step 1517 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

NEBNext Ultra II Ligation Module (# E7595)

Ligation of adapter to fragment

1xIDTE (10 mM Tris-HCl (pH 8.0) with 0.1 mM EDTA)

IDT

Sample library dilution

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

From one of suppliers:

QuantaBio

Beckman Coulter

Beckman Coulter

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