TITLE: SSLib v2.0 (Gansauge et al. 2017)
AUTHORS: Alicia Grealy

[DESCRIPTION]
This bench protocol is based on the work of Gansauge and Meyer (2013)  and Gansauge et al (2017), for preparing shotgun libraries from single-stranded DNA, typically for ancient and degraded DNA.


[BEFORE_START]
Note that you will need to adjust steps depending on the equipment you have (e.g., a Thermal shakers and hybridisation ovens can be used interchangably). If you only have heat blocks, you will need to keep beads moving by regular vortexing. For high-throughput, it may be advisable to use a 96-well plate magent coupled with 0.2 ml tubes rather than 1.5 ml tubes. 

Always include the positive control oligo CL104 in the library preparation, as well as a no-template control and any extraction controls. 

Note that CL78 should have a 5' phosphate, but this is missing in Gansauge and Meyer (2013).

Note that 0.1X BWT is NOT just a diluted version of 1X BWT. It only contains a tenth of the NaCl but is the same for most of the other reagents. Here, I call it WASH A as per Gansauge and Meyer (2013) to avoid confusion. 

I have adjusted the volumes of buffers to make only 10 ml of the wash buffers, which is typically enough for a batch of 12 libraries. If you are doing more reactions, scale up the recipe.

Note that some oligos need to be resuspended in a specific buffer. 

Note that the splinter oligo used here is not published in Gansauge et al. (2017), but was personally recommended to me by Matthias Meyer. 

Store PEG and ATP at -20 deg C and avoid repeated rounds of freeze thawing. 

Store MyOne C1 Streptavidin beads at 4 deg C in a fridge. 

[GUIDELINES]
Use at your own risk. You are responsible for double-checking that everything is correct!

Familiarise yourself with the relevent literature (Gansauge and Meyer 2013, and Gansauge et al. 2017) before attempting!


[STEPS]
SECTION: Preparation
1. 
 

"Suit up" in this order: hair net, nitrile gloves, facemask, coveralls, gumboots, booties, second pair of gloves.
SECTION: Preparation
3. Ensure ice is available. Thaw reagents on ice as needed. Keep enzymes on ice at all times. Do not vortex enzymes to mix but mix by flicking the tube gently. Pulse centrifuge all reagents before opening. 
SECTION: Preparation
4. Label tubes. 

 

SECTION: Preparation
5. Prepare all necessary buffers and UV decontaminate where appropriate. 
 

 

SECTION: Preparation
6. Before resuspending oligos, pulse centrifuge to collect the pellet at the bottom of the tube. Add the appropriate buffer (see Materials) and vortex thoroughly. Store at -20 deg C. Dilute out the working concentrations (below) and store at -20 deg C when not in use. Thaw on ice. Vortex and pulse centrifuge after each thaw. Before beginning library preparation, make sure you have enough of each working stock prepared!


 


 



SECTION: Preparation
7. Pre-program the thermal cycler and thermalshaker.
SECTION: Purify and ligate adapters
8. Combine the following in a 0.2 ml Lo-bind PCR tube. Vortex and pulse centrifuge. 

 

SECTION: Preparation
2. Prepare the space by decontaminating surfaces with 10% household bleach followed by 70% ethanol. UV irradiate pipettes and racks. Racks should be bleached between subsequent uses and UV irradiated. 
SECTION: Purify and ligate adapters
10. Incubate both reactions for 20 minutes at 37 deg C, followed by 1 min at 95 deg C in a thermal cycler with heated lid. 
SECTION: Purify and ligate adapters
9. Combine the following in a 0.2 ml Lo-bind PCR tube. Vortex and pulse centrifuge. 

 

SECTION: Purify and ligate adapters
11. Combine the following in a 0.2 ml Lo-bind PCR tube. Vortex and pulse centrifuge. This makes 10/20 uM of CL78/Splinter (DS1).

 

SECTION: Purify and ligate adapters
13. Incubate for 10 sec at 95 deg C, followed by a ramp down to 10 deg C at 0.1 deg C/sec in a thermal cycler with a heated lid. 
 

SECTION: Purify and ligate adapters
12. Combine the following in a 0.2 ml Lo-bind PCR tube. Vortex and pulse centrifuge. This makes 200 uM of CL53/CL73 (DS2).


 

SECTION: Dephosphorylation, heat denaturation, and ligation of first adapter
15. 
 

Set a heat block or thermal shaker to 45 deg C and set another thermal shaker to 35 deg C. 
SECTION: Purify and ligate adapters
14. Add 50 ul of TE buffer to DS2 to make 100 uM of DS2 (CL53/CL73). 
SECTION: Dephosphorylation, heat denaturation, and ligation of first adapter
16. Make up the following master mix in a 1.5 ml Lo-bind tube. Vortex and pulse centrifuge. 



 

SECTION: Dephosphorylation, heat denaturation, and ligation of first adapter
17. Aliquot 34 ul per reaction into a 0.2 ml Lo-bind PCR tube.
SECTION: Dephosphorylation, heat denaturation, and ligation of first adapter
18. To make the total reaction volume up to 46 ul, add:

Up to 12 ul DNA to each sample reaction. Make up the remainder with Ultrapure water. Typically input 3x10^8 - 3x10^11 double-stranded molecules; 1 fmol-1pmol single-stranded DNA; 13 pg-14 ng of ca. 40 bp DNA--this is typically 20% of the extract. 

1 ul of 0.1 uM CL104 positive control oligo to the positive conrol reaction + 11 ul Ultrapure water. We are inputing 3.01x10^10 molecules of single-stranded CL104 into the library preparation. 

12 ul of Ultrapure water to the no-template control reaction. 
SECTION: Dephosphorylation, heat denaturation, and ligation of first adapter
19. Incubate for 10 min at 37 deg C in a thermal cycler with a heated lid followed by 2 min at 95 deg C. Place immediately into an ice water bath. 
SECTION: Dephosphorylation, heat denaturation, and ligation of first adapter
20. Make up the following master mix in a 1.5 ml Lo-bind tube. Vortex and pulse centrifuge. 


 

SECTION: Dephosphorylation, heat denaturation, and ligation of first adapter
21. Aliquot 34.4 ul to the reactions from Step 19. Vortex and pulse centrifuge. 
SECTION: Dephosphorylation, heat denaturation, and ligation of first adapter
22. Incubate 1 hr at 37 deg C in a thermal cycler, followed by 1 min at 95 deg C. Transfer immediately to ice. 


 

SECTION: Dephosphorylation, heat denaturation, and ligation of first adapter
23. Pause point: Reactions can be frozen at -20 deg C for several days before proceeding. If you proceed immediately, skip Step 30 below. 
SECTION: Immobilisation of ligation products on bead
24. Allow MyOne C1 Streptavidin beads to come to room temperature. Vortex. 
SECTION: Immobilisation of ligation products on bead
25. For every sample (including controls) aliquot 20 ul of MyOne C1 Streptavidin beads to a 1.5 ml Lo-bind tube. Include 20 ul extra for pipetting error. (e.g., if you have 5 samples + 2 controls, aliquot 160 ul of beads). 

 

SECTION: Immobilisation of ligation products on bead
26. Allow the beads to separate from solution on a magnetic rack for 1-2 minutes. Discard the supernatant. Add 500 ul of Bead Binding Buffer. Vortex.


 

SECTION: Immobilisation of ligation products on bead
27. Repeat Step 26. 
SECTION: Immobilisation of ligation products on bead
28. Discard the supernatant. Resuspend beads in 250 ul Bead Binding Buffer per sample including controls and pipetting error (e.g., if you have 5 samples + 2 controls, resuspend the beads in 2 ml of Bead Binding Buffer). Pipette up and down gently to resuspend to avoid generating excessive bubbles. 
SECTION: Immobilisation of ligation products on bead
29. Aliquot 250 ul of bead suspension to new 1.5 ml Lo-bind Safelock tubes (1 per sample + controls). 
SECTION: Immobilisation of ligation products on bead
30. Incubate ligation reactions from Step 22 for 1 min at 95 deg C and transfer to an ice water bath for 2-5 min. 


 

SECTION: Immobilisation of ligation products on bead
31. Add the ligation reactions from Step 22 to the bead suspension. Vortex. 
SECTION: Immobilisation of ligation products on bead
32. Incubate for 20 min at room temperature with inversion. Pulse centrifuge. 

 

SECTION: Immobilisation of ligation products on bead
33. Pellet the beads with the magnetic rack and discard the supernatant. 
SECTION: Immobilisation of ligation products on bead
34. Add 200 ul of Wash A to each sample. Vortex. Pulse centrifuge. Pellet beads with a magnetic rack and discard the supernatant. 
SECTION: Immobilisation of ligation products on bead
35. Add 100 ul Stringency Wash Buffer. Vortex. Incubate 3 min at 45 deg C in a heat block (or thermal shaker), vortexing every 30 sec. Pulse centrifuge. Pellet beads with a magenetic rack and discard the supernatant. 

 

SECTION: Immobilisation of ligation products on bead
36. Set the heat block to 65 deg C so it has time to heat up before needed. 

 

SECTION: Immobilisation of ligation products on bead
37. Add 200 ul of Wash B to each sample. Vortex. Pulse centrifuge. Pellet beads with a magnetic rack and discard the supernatant. 
SECTION: Primer annealing and extension
38. Make up the following master mix in a 1.5 ml Lo-bind tube. Vortex and pulse centrifuge. 



 

SECTION: Primer annealing and extension
39. Add 48 ul of the master mix to each reaction from Step 37. Vortex and pulse centrifuge. 
SECTION: Primer annealing and extension
40. Incubate 2 min at 65 deg C in a heat block (or thermal shaker), and place immediately in an ice water bath for 2-5 min. Transfer the rack to room temperature. 
SECTION: Primer annealing and extension
41. Set the heat block back to 45 deg C so that it has time to cool down before needed. 
SECTION: Primer annealing and extension
42. Add 2 ul of Klenow fragment (10 U/ul) to the reactions from Step 40. Vortex and pulse centrifuge. 
SECTION: Primer annealing and extension
43. Incubate 5 min at 25 deg C (or room temperature), followed by 25 min at 35 deg C in a thermo shaker, with shaking at 1000 rpm. Do not allow beads to settle. 
SECTION: Primer annealing and extension
44. Remove the reactions and pellet the beads on a magnetic rack. Set the thermo shaker to 22 deg C. 
SECTION: Post-extension washes
45. Add 200 ul of Wash A to each sample. Vortex. Pulse centrifuge. Pellet beads with a magnetic rack and discard the supernatant. 
SECTION: Post-extension washes
46. Add 100 ul Stringency Wash Buffer. Vortex. Incubate 3 min at 45 deg C in a heat block (or thermal shaker), vortexing every 30 sec. Pulse centrifuge. Pellet beads with a magenetic rack and discard the supernatant. 
SECTION: Post-extension washes
47. Add 200 ul of Wash B to each sample. Vortex. Pulse centrifuge. Pellet beads with a magnetic rack and discard the supernatant. 
SECTION: Ligation of second adapter
48. Make up the following master mix in a 1.5 ml Lo-bind tube. Vortex and pulse centrifuge. 


 

SECTION: Ligation of second adapter
49. Add 100 ul of master mix to each tube. Vortex and pulse centrifuge. 
SECTION: Ligation of second adapter
50. Incubate for 1 hr at 22 deg C in the thermo shaker, shaking at 1000 rpm. 

 

SECTION: Ligation of second adapter
51. Remove samples and set shaker to 95 deg C. Pellet the beads in a magnetic rack and discard the supernatant.
SECTION: Post-ligation washes
52. Add 200 ul of Wash A to each sample. Vortex. Pulse centrifuge. Pellet beads with a magnetic rack and discard the supernatant. 
SECTION: Post-ligation washes
53. Add 100 ul Stringency Wash Buffer. Vortex. Incubate 3 min at 45 deg C in a heat block (or thermal shaker), vortexing every 30 sec. Pulse centrifuge. Pellet beads with a magenetic rack and discard the supernatant. 
SECTION: Post-ligation washes
54. Add 200 ul of Wash B to each sample. Vortex. Pulse centrifuge. Pellet beads with a magnetic rack and discard the supernatant. 
SECTION: Elution of the final library
55. Add 25 ul EBT buffer to each sample. Vortex and pulse centrifuge.
SECTION: Elution of the final library
56. Incubate for 2 min at 95 deg C in a thermo shaker (without shaking). 
SECTION: Elution of the final library
57. Transfer tubes immediately to the magnetic rack and pellet the beads. Transfer the supernatant to a clean 0.5 ml Lo-bind tube.
SECTION: Elution of the final library
59. Libraries can be stored at -20 deg C until amplification. For long-term storage, store at -80 deg C. 
SECTION: Quant the library
60. Make up the following master mix in a 1.5 ml Lo-bind tube. Vortex and pulse centrifuge. 



 

SECTION: Quant the library
61. Make up the following master mix in a 1.5 ml Lo-bind tube. Vortex and pulse centrifuge. 

 

SECTION: Quant the library
63. Add 1 ul of DNA sample to the corresponding PCR tubes according to the scheme below. Pulse centrifuge the tubes.
 

 

SECTION: Quant the library
62. Add 24 ul of master mix to the corresponding PCR tubes. Pulse centrifuge the tubes. 
SECTION: Elution of the final library
58. Create a 1in20 dilution of each library in Ultrapure water (1 ul library + 19 ul Ultrapure water). Vortex and pulse centrifuge. 
SECTION: Quant the library
64. Take the strip tubes to a post-PCR space. Place in thermal cycler and run the following program:

        95 deg C for 10 min

        Followed by 50 cycles of:
        
        95 deg C for 30 sec 
        60 deg C for 30 sec
        72 deg C for 30 sec
SECTION: Quant the library
66. Use the CT values from the qPCR to generate a standard curve for the standards in order to calculate how many template copies are present in each library. The positive control is used to calculate the efficiency of the library prep:

(# Copies of CL104 from Assay A / # copies CL104 from Assay B) * 100


 


This assay is also used to determine the number of cycles to give the indexing PCR, which needs to be stopped during the linear phase. See my library amplification protocol to proceed with the next step. 
SECTION: Quant the library
65. Electrophorese 10 ul of the PCR product from the libraries (not standards) and controls on a 2% agarose gel. 


 

SECTION: Index/amplify the library
67. Make up the following master mix in a 1.5 ml Lo-bind tube. Ensure to prepare enough master mix for 4 reactions per library plus pipetting error. Vortex and pulse centrifuge. 

 

 


 


SECTION: Index/amplify the library
68. Add 19 ul of master mix to the corresponding PCR tubes. Pulse centrifuge the tubes. 
SECTION: Index/amplify the library
69. Add  0.5 ul of the corresponding forward indexing primer to the appropriate reaction tube. Pulse centrifuge the tubes. 
SECTION: Index/amplify the library
70. Add 0.5 ul of the corresponding reverse indexing primer to the appropriate reaction tube. Pulse centrifuge the tubes. 
SECTION: Index/amplify the library
71. Add 5 ul of DNA sample to the corresponding reaction tubes according to the scheme below. Pulse centrifuge the tubes.

e.g., 
 

 

SECTION: Index/amplify the library
72. Take the strip tubes to a post-PCR space. Place in qPCR machine and run the following program:

        95 deg C for 10 min
    
     Followed by _________ cycles of:
        
        95 deg C for 30 sec 
        60 deg C for 30 sec
        72 deg C for 30 sec

 

SECTION: Purify the libraries
73. Pulse centrifuge the PCR tubes. Combine replicate PCR reactions into a 1.5 ml Lo-bine Safelock tube. Vortex and pulse centrifuge.
SECTION: Purify the libraries
74. Purify the libraries using SeraMag Speed Beads or SeraMag Select using a 1.6X beads : reaction volume (i.e., 160 ul). Follow the guidelines below:

https://www.gelifesciences.co.jp/catalog/pdf/SeraMagSelect_UserGuide.pdf

Elute in 35 ul of Ultrapure water. 
SECTION: Quantitate the libraries
75. Dilute the libraries 1 in 10 in Ultrapure water (i.e., 1 ul library in 9 ul Ultrapure water). 
SECTION: Quantitate the libraries
76. Use a LabChip GXII or equivalent fragment analyser (HiSense kit) to measure the molarity of the libraries between 160-500 bp. 

https://www.perkinelmer.com/Content/LST_Software_Downloads/LabChip_GX_User_Manual.pdf

 

SECTION: Pool libraries
77. Pool libraries in equimolar concentrations such that the total amount of DNA per library does not exceed 500-1000 ng. 
 

SECTION: Pool libraries
78. Use a Vivaspin 500 (MWCO 30,000 Da) centrifugal column to concentrate each library to 20-40 ul. Centrifuge at 15,000 rcf with the membrane facing outwards for 30 sec at a time. 

https://www.sartorius.com/shop/ww/en/usd/applications-laboratory-filtration-ultrafiltration/vivaspin-500%2C-30%2C000-mwco-pes%2C-25pc/p/VS0121

Alternatively, concentrate the libraries using a SpeedyVac system, following the manufacturer's instructions. 

https://assets.thermofisher.com/TFS-Assets/LED/manuals/DNA130%20User%20Manual%20final%20versionpdf.pdf
SECTION: Size select and purify
79. Run each pool in duplicate across two lanes (20 ul each) of a PippinHT electrophoresis system (2% gel, Marker 20B), selecting fragments between 160-500 bp and following the manufacturer's instructions:

http://www.sagescience.com/wp-content/uploads/2015/10/PippinHT-Operations-Manual-Rev-B_460005.pdf
SECTION: Size select and purify
80. Combine replicates. Purify the libraries using SeraMag Speed Beads or SeraMag Select using a 2X beads : reaction volume (i.e., 160 ul). Follow the guidelines below:

https://www.gelifesciences.co.jp/catalog/pdf/SeraMagSelect_UserGuide.pdf

Elute in 25 ul of Ultrapure water. 
SECTION: Quantitate the final library
81. Dilute the libraries 1/2, 1/5, 1/10  in Ultrapure water (i.e., create a serial dilution in 10 ul volume).
SECTION: Quantitate the final library
82. Measure the concentration of the neat library and these dilutions in duplicate on the Qubit following the manufacturer's instructions. 

https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017209_Qubit_4_Fluorometer_UG.pdf
SECTION: Quantitate the final library
83. Measure the molarity of the neat library and dilutions on a LabChip GXII Hisense kit (or equivalent fragment analyser) following the manufacturer's instructions:

https://www.perkinelmer.com/Content/LST_Software_Downloads/LabChip_GX_User_Manual.pdf
SECTION: Quantitate the final library
84. Based on the average fragment length and Qubit measurement, calculate the molarity of the library dilutions. Create a standard curve to check that the concentrations are linear. If they can be "trusted", extrapolate the neat concentration based on the dilutions. Average all the measurements of the neat concentration to get the best estimate of the library molarity.
SECTION: Sequencing
85. Dilute the library to between 2-4 nM in Ultrapure water. 
 

SECTION: Sequencing
86. Follow the manufacturer's instructions to perform the sequencnig run on your platform of choice.