Library preparation (dsDNA single indexing, full-UDG, no split)
Marcel Keller, Christiana L Scheib
Abstract
Protocol for the preparation of single indexed double-stranded DNA libraries for Illumina sequencing, optimized for ultra-short ancient DNA molecules, modified from Meyer & Kircher (2010) Cold Spring Harb. Protoc. (doi: 10.1101/pdb.prot5448). This protocol does include treatment with UDG (USER) to remove DNA damage in form of deaminated cytosines.
Before start
Previous step:
This protocol follows the extract purification protocol.
Following step:
This protocol ends with the PCR setup. Proceed with the PCR and library purification protocol.
Preparations:
Get the following consumables and equipment:
| A | B |
|---|---|
| Numbers | Equipment and consumables |
| 1 | 0.2 ml tube rack |
| 2 | 1.5 ml tube rack |
| 1 | 1.5 ml cool block |
| 1 | 0.2 ml cool block |
| 10 µl filter tips | |
| 20 µl filter tips | |
| 100 µl filter tips | |
| 200 µl filter tips | |
| 1000 µl filter tips | |
| [# of samples]×4 (tubes) | 0.2 µl PCR strips (8 tubes) |
| [# of samples]×2+4 | 1.5 ml tubes |
| 1 | 5 ml tube |
| [# of samples]×2 | MinElute columns |
| 1 | 50 ml Falcon (waste) |
Steps
Preparation
Turn the hood on full power and open the glass.
Spray hood and table bench surfaces with DNA Exitus, let sit a minute and wipe down with paper towels.
Wipe down outside surfaces of reagents/tips with DNA Exitus and place in the hood.
Label the following tubes:
5×1.5 ml tubes: ER, AL, FI, EB-1, EB-2
1×5 ml tube: PCR
0.2 ml PCR strips: ER, AL, FI, PCR
Label the 50 ml waste tube, PB tube and PE tube.
Aliquot EB buffer in tubes EB-1 and EB-2: each [# of samples]x30 µl plus 10%
Aliquot water for Master Mixes:
ER: [# of samples]×25.85 µl plus 10%
FI: [# of samples]×12.20 µl plus 10%
PCR: [# of samples]×17.00 µl plus 10%
Aliquot PB buffer to a 50 ml tube: [# of samples]x1000 µl plus 10%
Prepare PE (wash) buffer by adding ethanol.
Aliquot PE buffer to a 50 ml tube: [# of samples]x1380 µl plus 10%
Take DNA extracts out of the freezer to thaw at room temperature. Change gloves.
Blunt End Repair 1
Use 1.5 ml ER tube to set up the Blunt End Repair Master Mix On ice .
| A | B | C | D | E | F |
|---|---|---|---|---|---|
| REAGENT | STOCK | FINAL | UNIT | 1× VOL (µl) | NOTE |
| Water [already added] | 25.85 | ||||
| NEB Buffer 2 | 10 | 1.0 | X | 7.50 | vortex |
| dNTPs | 25 | 0.30 | mM | 0.90 | vortex |
| BSA | 20 | 0.20 | mg/ml | 0.75 | vortex |
| ATP | 10 | 1.00 | mM | 7.50 | vortex |
| T4 PNK | 10 | 0.40 | U/µl | 3.00 | on ice |
| USER enzyme | 1 | 0.06 | U/µl | 4.50 | on ice |
| Master Mix total | 50.00 | ||||
| Template DNA or water | 25.00 | ||||
| REACTION TOTAL | 75.00 |
Calculate +10% for all Master Mix components.
Add 20 µl Master Mix to each tube of the ER strip.
Vortex and spin down DNA extracts, add 25 µl of template DNA or water to each tube.
Mix carefully by resuspending, remove bubbles and spin down.
Incubate at 37°C for 3h 0m 0s.
Blunt End Repair 2
Add 3 µl T4 pol to each reaction.
| A | B | C | D | E | F |
|---|---|---|---|---|---|
| REAGENT | STOCK | FINAL | UNIT | 1× VOL (µl) | NOTE |
| T4 DNA Polymerase | 3 | 0.038 | U/µl | 3.00 | on ice |
| REACTION TOTAL | 78.00 |
Incubate at 25°C for 0h 30m 0s, then at 10°C for 0h 5m 0s
MinElute Purification 1
Take MinElute columns out of the fridge.
Turn on heat block 37°C for elution.
Label columns and tubes with sample ID numbers.
Add 500 µl PB buffer (binding buffer) to MinElute column. You can use the same tip.
Add end-repair reaction mix to the PB buffer inside the MinElute columns and mix by resuspending.
Spin at 13000rpm, discard supernatant, change gloves.
Add 690 µl PE buffer (wash buffer), change tip for every sample.
Spin at 13000rpm, discard supernatant, change gloves.
Spin at 13000rpm (dry spin).
Put column in new tube, change gloves.
Elute in 30 µl EB buffer (elution buffer), change tip for every sample.
Incubate at 37°C for 0h 10m 0s .
Spin at 13000rpm .
Discard the silica column and close the lid.
Adapter ligation
Use 1.5 ml AL tube to set up the Adapter Ligation Master Mix On ice.
| A | B | C | D | E | F |
|---|---|---|---|---|---|
| REAGENT | STOCK | FINAL | UNIT | 1× VOL (µl) | NOTE |
| Quick Ligation Buffer | 5.0 | 1.00 | × | 10 | vortex |
| Adapter Mix | 2.5 | 0.25 | µM | 5 | vortex |
| End Repair Enzyme Mix | 1.0 | 0.10 | U | 5 | on ice |
| Master Mix total | 20.00 | ||||
| Template DNA or water | 30.00 | ||||
| REACTION TOTAL | 50.00 |
Calculate +20% for all Master Mix components.
Add 20 µl of Master Mix to each tube of the AL strip.
Add 30 µl of end-repaired template DNA or water to each tube.
Incubate at 20°C for 0h 15m 0s.
MinElute purification 2
Take MinElute columns out of the fridge.
Turn on heat block 37°C for elution if not turned on already.
Label columns and tubes with sample ID numbers.
Add 500 µl PB buffer (binding buffer) to MinElute columns. You can use the same tip.
Add adapter ligation reaction mix to the PB buffer inside the MinElute columns and mix by resuspending.
Spin at 13000rpm, discard supernatant, change gloves.
Add 690 µl PE buffer (wash buffer), change tip for every sample.
Spin at 13000rpm, discard supernatant, change gloves.
Spin at 13000rpm (dry spin).
Put column in new tube, change gloves.
Elute in 30 µl EB buffer (elution buffer), change tip for every sample.
Incubate at 37°C for 0h 10m 0s.
Spin at 13000rpm.
Discard the silica column, close the lid.
Fill-in reaction
Use 1.5 ml FI tube to set up the Adapter Ligation Master Mix On ice.
| A | B | C | D | E | F |
|---|---|---|---|---|---|
| REAGENT | STOCK | FINAL | UNIT | 1× VOL (µl) | NOTE |
| Water [already added] | 12.2 | ||||
| Thermopol Buffer | 10 | 1.00 | × | 5.0 | vortex |
| dNTPs | 25 | 0.40 | mM | 0.8 | vortex |
| Bst polymerase | 8 | 0.32 | U/µl | 2.0 | on ice |
| Master Mix total | 20 | ||||
| Template DNA or water | 30 | ||||
| REACTION TOTAL | 50 |
Calculate +20% for all Master Mix components.
Add 20 µl Master Mix to each tube of the FI strip.
Add 30 µl of adapter-ligated template DNA or water to each tube.
Incubate at 37°C for 0h 30m 0s , then at 80°C for 0h 20m 0s.
Library amplification (PCR)
Use 5 ml PCR tube to set up PCR Master Mix On ice.
| A | B | C | D | E | F |
|---|---|---|---|---|---|
| REAGENTS | STOCK | FINAL | UNIT | 1× VOL (µl) | NOTE |
| Water [already added] | 17 | ||||
| 10x PCR buffer | 10 | 1.00 | X | 10 | vortex |
| MgCl2 | 25 | 2.50 | mM | 10 | vortex |
| BSA | 20 | 1.00 | mg/ml | 5 | vortex |
| dNTPs 10 mM | 40 | 0.80 | mM | 2 | vortex |
| HGS Taq Diamond | 1 | 0.02 | U | 2 | on ice |
| Universal primer 1.0 | 10 | 0.2 | µM | 2 | vortex |
| Master Mix total | 48 | ||||
| Indexing primer | 10 | 0.2 | µM | 2 | vortex |
| Template DNA or water | 50 | ||||
| REACTION TOTAL | 100 |
Calculate +10% for all Master Mix components.
Aliquot 48 µl of Master Mix to the tubes of the PCR strip.
Vortex indexes slightly and spin them down with a table centrifuge.
Add 2 µl of the indexing primer (10 µM) to the respective tube with Master Mix.
Add 50 µl of adapter fill-in reaction mix or water to the respective tube with Master Mix. Mix by resuspending with the pipet.
Check that the lids are tightly sealed and take the strips to the modern lab for PCR.
