TITLE: Library preparation (dsDNA double indexing, full-UDG, 2x split)
AUTHORS: Marcel Keller, Christiana L Scheib

[DESCRIPTION]
Protocol for the preparation of double 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:

 


[GUIDELINES]
Please read the general guidelines for working in the Ancient DNA protocol collection – University of Tartu, Institute of Genomics.

[STEPS]
SECTION: Blunt End Repair 1
11. Use 1.5 ml ER tube to set up the Blunt End Repair Master Mix on ice .

 

SECTION: Blunt End Repair 1
12. Add 20 µl Master Mix to each tube of the ER strip.
SECTION: Blunt End Repair 1
13. Vortex and spin down DNA extracts, add 25 µl of template DNA or water to each tube.
SECTION: Blunt End Repair 1
14. Mix carefully by resuspending, remove bubbles and spin down.
SECTION: Blunt End Repair 1
15. Incubate at 37 °C for 180 min.

 

SECTION: MinElute Purification 1
18. Take MinElute columns out of the fridge.
SECTION: MinElute Purification 1
19. Turn on heat block 37 °C  for elution.

SECTION: MinElute Purification 1
20. Label columns and tubes with sample ID numbers.
SECTION: MinElute Purification 1
21. Add 500 µl PB buffer (binding buffer) to MinElute column. You can use the same tip.
SECTION: MinElute Purification 1
23. Spin at 13000 rpm, 1 min, discard supernatant, change gloves.

SECTION: MinElute Purification 1
24. Add 690 µl PE buffer (wash buffer), change tip for every sample.
SECTION: MinElute Purification 1
25. Spin at 13000 rpm, 1 min, discard supernatant, change gloves.
SECTION: MinElute Purification 1
26. Spin at 13000 rpm, 1 min (dry spin).
SECTION: MinElute Purification 1
27. Put column in new tube, change gloves.
SECTION: MinElute Purification 1
28. Elute in 30 µl EB buffer (elution buffer), change tip for every sample.
SECTION: Adapter ligation
33. Use 1.5 ml AL tube to set up the Adapter Ligation Master Mix on ice.
 
 

SECTION: Adapter ligation
34. Add 20 µl of Master Mix to each tube of the AL strip.
SECTION: Adapter ligation
35. Add 30 µl of end-repaired template DNA or water to each tube.
SECTION: Adapter ligation
36. Incubate at 20 °C for 15 min.


SECTION: MinElute Purification 1
30. Spin at 13000 rpm, 2 min .



SECTION: MinElute purification 2
37. Take MinElute columns out of the fridge.
SECTION: MinElute purification 2
38. Turn on heat block 37 °C  for elution if not turned on already.

SECTION: MinElute purification 2
39. Label columns and tubes with sample ID numbers.
SECTION: MinElute purification 2
40. Add 500 µl PB buffer (binding buffer) to MinElute columns. You can use the same tip.
SECTION: MinElute purification 2
42. Spin at 13000 rpm, 1 min, discard supernatant, change gloves.

SECTION: MinElute purification 2
43. Add 690 µl PE buffer (wash buffer), change tip for every sample.
SECTION: MinElute purification 2
44. Spin at 13000 rpm, 1 min, discard supernatant, change gloves.
SECTION: MinElute purification 2
45. Spin at 13000 rpm, 1 min (dry spin).
SECTION: MinElute purification 2
46. Put column in new tube, change gloves.
SECTION: MinElute purification 2
47. Elute in 30 µl EB buffer (elution buffer), change tip for every sample.
SECTION: MinElute purification 2
49. Spin at 13000 rpm, 2 min.

SECTION: Fill-in reaction
52. Use 1.5 ml FI tube to set up the Adapter Ligation Master Mix on ice.

 

 
SECTION: Fill-in reaction
53. Add 20 µl Master Mix to each tube of the FI strip.
SECTION: Fill-in reaction
54. Add 30 µl of adapter-ligated template DNA or water to each tube.
SECTION: Fill-in reaction
55. Incubate at 37 °C for 30 min , then at 80 °C for 20 min.

 

SECTION: Library amplification (PCR)
56. Use 5 ml PCR tube to set up PCR Master Mix on ice.

 



 

SECTION: Library amplification (PCR)
57. Aliquot 142 µl of Master Mix to the tubes of the PCR-A strip.
SECTION: Library amplification (PCR)
58. Vortex indexes slightly and spin them down with a table centrifuge.
SECTION: Library amplification (PCR)
59. Add 4 µl of i7 indexing primer (10 µM) to the respective tube with Master Mix.
SECTION: Library amplification (PCR)
61. Add 50 µl of adapter fill-in reaction mix or water to the respective tube with Master Mix.
SECTION: Library amplification (PCR)
63. Check that the lids are tightly sealed and take the strips to the modern lab for PCR.
SECTION: MinElute Purification 1
32. 
 

SECTION: MinElute purification 2
51. 
 

SECTION: MinElute Purification 1
22. Add end-repair reaction mix to the PB buffer inside the MinElute columns and mix by resuspending.
SECTION: MinElute Purification 1
29.  Incubate at 37 °C for 10 min .
66. 
SECTION: MinElute Purification 1
31. Discard the silica column and close the lid.
SECTION: MinElute purification 2
41. Add adapter ligation reaction mix to the PB buffer inside the MinElute columns and mix by resuspending.
SECTION: MinElute purification 2
48. Incubate at 37 °C for 10 min.

SECTION: MinElute purification 2
50. Discard the silica column, close the lid.
SECTION: Preparation
1. Turn the hood on full power and open the glass.
SECTION: Preparation
2. Spray hood and table bench surfaces with DNA Exitus, let sit a minute and wipe down with paper towels.
SECTION: Preparation
3. Wipe down outside surfaces of reagents/tips with DNA Exitus and place in the hood. 
SECTION: Preparation
4. 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-A, PCR-B
Label the 50 ml waste tube, PB tube and PE tube.
SECTION: Preparation
5. 
Aliquot EB buffer in tubes EB-1 and EB-2: each [# of samples]x30 µl plus 10%
SECTION: Preparation
6. Aliquot water for Master Mixes:
ER:  [# of samples]×25.85 µl plus 10%
FI: [# of samples]×12.20 µl plus 10%
PCR:  [# of samples]×84.00 µl plus 10%
SECTION: Preparation
8. Prepare PE (wash) buffer by adding ethanol.
SECTION: Blunt End Repair 2
16. Add 3 µl T4 pol to each reaction.

 

SECTION: Blunt End Repair 2
17. Incubate at 25 °C for 30 min, then at 10 °C for 5 min 

 

SECTION: Preparation
7. Aliquot PB buffer to a 50 ml tube: [# of samples]x1000 µl plus 10%
SECTION: Preparation
9. Aliquot PE buffer to a 50 ml tube:  [# of samples]x1380 µl plus 10%
SECTION: Preparation
10. Take DNA extracts out of the freezer to thaw at room temperature. Change gloves.
SECTION: Library amplification (PCR)
60. Add 4 µl of i5 indexing primer (10 µM) to the respective tube with Master Mix.
SECTION: Library amplification (PCR)
62. Mix by resuspending and pipet 100 µl of the reaction to the PCR-B strip tubes.