TITLE: hyRAD (Suchan et al. 2016; Grealy et al.) 
AUTHORS: Alicia  Grealy

[DESCRIPTION]
This bench protocol is based on the work of Tomasz Suchan, for performing hyRAD with RNA baits, with some changes. 

[BEFORE_START]
Read Suchan et al. (2016) 
Also see:  dx.doi.org/10.17504/protocols.io.mt2c6qe by George Olah

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 (Suchan et al. 2016, Peterson et al. ) before attempting!

[STEPS]
SECTION: Probe synthesis
1. Preparation
SECTION: Probe synthesis
2. Enzymatic digestion
SECTION: Probe synthesis
1.1. 
 

"Suit up" in this order: hair net, nitrile gloves, facemask, coveralls, gumboots, booties, second pair of gloves.
SECTION: Probe synthesis
3. Adapter ligation
SECTION: Probe synthesis
1.2. 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: Probe synthesis
1.3. 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: Probe synthesis
1.4. Label tubes. 

 
 

 
 
SECTION: Probe synthesis
1.5. Prepare all necessary buffers and UV decontaminate where appropriate. 
 

 
 
 


 
SECTION: Probe synthesis
1.6. 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: Probe synthesis
1.7. Pre-program the thermal cycler.
SECTION: Probe synthesis
1.8. Combine the following in a 0.2 ml Lo-bind PCR tube. Vortex and pulse centrifuge. 

 

SECTION: Probe synthesis
1.9. Combine the following in a 0.2 ml Lo-bind PCR tube. Vortex and pulse centrifuge. 

 
 
 

 
SECTION: Probe synthesis
1.10. Incubate in a thermal cycler at:
        
       95 deg C for 1 min
       Cool at a rate of 0.1 deg C / sec until the solution reaches 20 deg C

Store at -20 deg C. 
SECTION: Probe synthesis
2.1. Label tubes. 

 
 
 

 
SECTION: Probe synthesis
2.2. Combine the following in a 1.5 ml Lo-bind Safelock tube. Vortex and pulse centrifuge. 
 
 
 

 
 

SECTION: Probe synthesis
2.3. Add 40 ul of master mix to 10 ul of DNA (at 100 ng/ul ca. 1000 ng total) in a 0.2 ml Lo-bind PCR tube. Vortex and pulse centrifuge. 
 

 

 

SECTION: Probe synthesis
2.4. Incubate in a thermal cycler at:
        
       37 deg C for 4 hr
       Hold at 4 deg C
SECTION: Probe synthesis
2.5. Combine any replicates.     
SECTION: Probe synthesis
3.1. Label tubes. 
 
 

SECTION: Probe synthesis
2.6. SPRI bead clean-up

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 20 ul of 10 mM Tris-HCl. 
 

SECTION: Probe synthesis
2.7. Qubit

Measure the concentration using the Qubit HS kit following the manufacturer's instructions. 

https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017209_Qubit_4_Fluorometer_UG.pdf
 

SECTION: Probe synthesis
2.8. Agarose gel electrophoresis

Electrophorese 2 ul of product (200-500 ng in 5-10 ul) on a 2% agarose gel. 

 

 

SECTION: Probe synthesis
3.2. Combine the following in a 1.5 ml Lo-bind Safelock tube. Vortex and pulse centrifuge. 
 
 

 

SECTION: Probe synthesis
3.3. Add 11 ul of the above master mix to 9 ul of digested DNA in a 0.2 ml Lo-bind PCR tube. Vortex and pulse centrifuge.
SECTION: Probe synthesis
3.4. Incubate in a thermal cycler at:
        
       16 deg C for ca. 20 hr (16 hr - overnight)

Combine any replicates.
 

SECTION: Probe synthesis
3.5. SPRI bead clean-up

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 21 ul of 10 mM Tris-HCl. 
 

SECTION: Probe synthesis
3.6. Qubit

Measure the concentration using the Qubit HS kit following the manufacturer's instructions. 

https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017209_Qubit_4_Fluorometer_UG.pdf
 

SECTION: Probe synthesis
4. qPCR quant the RAD library

 

SECTION: Probe synthesis
5. Size selection using Pippin HT
 

SECTION: Probe synthesis
5.1. 
Depending on the total yield and concentration determined using the Qubit (see Step 3.6 above), run approximately 1 ug of DNA in 20 ul (ca. 75 ng/ul) of each samples across a lane of a PippinHT electrophoresis system (2% gel, Marker 20B), selecting fragments 272 bp in size (272 bp peak with tight range 212-332 bp--this equates to an insert size of 180 bp) and following the manufacturer's instructions:

http://www.sagescience.com/wp-content/uploads/2015/10/PippinHT-Operations-Manual-Rev-B_460005.pdf

 

SECTION: Probe synthesis
4.1. Label tubes. 
 
 


SECTION: Probe synthesis
4.2. Combine the following in a 1.5 ml Lo-bind Safelock tube. Vortex and pulse centrifuge. 
 
 

SECTION: Probe synthesis
4.3. Add 24 ul of master mix to the corresponding PCR tubes. Pulse centrifuge the tubes. 
SECTION: Probe synthesis
4.5. Add 1 ul of DNA sample to the corresponding PCR tubes according to the scheme below. Pulse centrifuge the tubes.


 e.g.,
 
 

SECTION: Probe synthesis
4.4. Dilute each library 1 in 20 (i.e., 1 ul of library in 19 ul of Ultrapure water). Vortex and pulse centrifuge. 
SECTION: Probe synthesis
4.6. 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: Probe synthesis
4.7. Agarose gel electrophoresis

Electrophorese 2 ul of product (200-500 ng in 5-10 ul) on a 2% agarose gel. 

 

 

SECTION: Probe synthesis
5.2.  SPRI cleanup

Combine any replicates. Purify the libraries using SeraMag Speed Beads or SeraMag Select using a 2X beads : reaction volume. Follow the guidelines below:

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

Elute in 21 ul of Ultrapure water. 
 

SECTION: Probe synthesis
5.3. Qubit

Measure the concentration using the Qubit HS kit following the manufacturer's instructions. 

https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017209_Qubit_4_Fluorometer_UG.pdf
 

SECTION: Probe synthesis
6. qPCR quant the size-selected RAD library

 

Follow Steps 4.1 - 4.7 above to perform the qPCR if so desired. 
 

SECTION: Probe synthesis
7. Index / amplify the RAD library


SECTION: Probe synthesis
7.1. Label tubes. 
 
 

SECTION: Probe synthesis
7.2. Combine the following in a 1.5 ml Lo-bind Safelock tube. Vortex and pulse centrifuge. Ensure to prepare enough master mix for multiple reactions per library plus pipetting error. 
 

 

 

 


 
 

SECTION: Probe synthesis
7.3. Add 41 ul of master mix to the corresponding PCR tubes. Pulse centrifuge the tubes. 
SECTION: Probe synthesis
7.6. Add 5 ul of purified size-selected RAD library  to the corresponding PCR tubes according to the scheme below. Pulse centrifuge the tubes.

 


 e.g.,
 
 

 
 
SECTION: Probe synthesis
7.7. Take the strip tubes to a post-PCR space. Place in thermal cycler and run the following program:

        98 deg C for 10 min

        Followed by 30 cycles of:
        
        985 deg C for 20 sec 
        60 deg C for 30 sec
        72 deg C for 40 sec

        Then a final extension of:

        72 deg C for 10 min
SECTION: Probe synthesis
7.8. Pool replicate reactions into a 1.5 ml Lo-bind Safelock tube. Vortex and pulse centrifuge. 
SECTION: Probe synthesis
7.9. SPRI bead clean-up

Purify the libraries using SeraMag Speed Beads or SeraMag Select using a1.4X beads : reaction volume. Follow the guidelines below:

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

Elute in 40 ul of Ultrapure water. 
 

SECTION: Probe synthesis
7.4. Add  2 ul of the corresponding forward indexing primer to the appropriate reaction tube. Pulse centrifuge the tubes. 
SECTION: Probe synthesis
7.5. Add  2 ul of the corresponding forward indexing primer to the appropriate reaction tube. Pulse centrifuge the tubes. 
SECTION: Probe synthesis
7.10. Qubit

Measure the concentration using the Qubit HS kit following the manufacturer's instructions. 

https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017209_Qubit_4_Fluorometer_UG.pdf
 

SECTION: Probe synthesis
7.11. Agarose gel electrophoresis

Electrophorese 5 ul of product (200-500 ng in 5-10 ul) on a 2% agarose gel. 

 

 

 

SECTION: Probe synthesis
8. Pool amplified libraries
SECTION: Probe synthesis
7.12. Use a LabChip GXII or equivalent fragment analyser (HiSense kit) to measure the molarity of the libraries. 

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

Alternatively, use the Qubit concentration and the average fragment size seen on the gel to estimate the molarity of the libraries:

http://www.endmemo.com/bio/dnamolarity.php
SECTION: Probe synthesis
8.1. Label tubes. 
 
 
 
 

 
 
SECTION: Probe synthesis
8.2. Pool libraries in equimolar concentrations such that the total volume is is approximately 100 ul. 
 

SECTION: Probe synthesis
8.3. Aliquot 20 ul of the pooled RAD library into a new 1.5 ml Lo-bind Safelock tube for future sequencing. Store at -20 deg C. See Steps 58-63 in the following protocol to quantitate and sequence the final RAD library. 


SECTION: Probe synthesis
9. Adapter removal
SECTION: Probe synthesis
9.1. Label tubes. 
 
 

SECTION: Probe synthesis
9.2. Combine the following in a 1.5 ml Lo-bind Safelock tube. Vortex and pulse centrifuge. 
 
 
 

 

SECTION: Probe synthesis
9.3. Aliquot 31.4 ul of the above master mix into 0.2 ml Lo-bind PCR tubes. 
SECTION: Probe synthesis
9.4. Add 18.6 ul (1 ug) of pooled purified RAD library to each tube. Vortex and pulse centrifuge. 
 

SECTION: Probe synthesis
9.5. Incubate the reactions in a thermal cycler at:

37 deg C for 4 hr

Combine the replicates. 
SECTION: Probe synthesis
9.6. SPRI bead clean-up

Purify the libraries using SeraMag Speed Beads or SeraMag Select using a 2X beads : reaction volume. Follow the guidelines below:

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

Elute in 30 ul of Ultrapure water. 
 

SECTION: Probe synthesis
9.7. Qubit

Measure the concentration using the Qubit HS kit following the manufacturer's instructions. 

https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017209_Qubit_4_Fluorometer_UG.pdf
 

SECTION: Probe synthesis
9.8. Agarose gel electrophoresis

Electrophorese 5 ul of product (200-500 ng in 5-10 ul) on a 2% agarose gel. 

 

 

SECTION: Probe synthesis
10. In-vitro transcription and biotinylation
SECTION: Probe synthesis
10.1. Label tubes. 
 
 
 
 

SECTION: Probe synthesis
10.2. Aliquot 10 ul of TURBO DNAse and 10 ul of SuperaseIn RNAse inhibitor into separate 0.2 ml Lo-bind tubes.
SECTION: Probe synthesis
10.3. Combine the following in a 1.5 ml Lo-bind Safelock tube. Vortex and pulse centrifuge. 
 
 
 

 
 
 

 

SECTION: Probe synthesis
10.4. Aliquot 13.5 ul of the above mater mix to 0.2 ml Lo-bind PCR tubes. Bring all tubes to a post-PCR space, including teh aliquots made in Step 10.2.
SECTION: Probe synthesis
10.5. Concentrate the "probe set" (i.e., the pool RAD library with the adapters cut off) such that roughly 500 ng in 6.5 ul can be added per reaction using a SpeedVac, following the manufacturer's instructions:

https://assets.thermofisher.com/TFS-Assets/LED/manuals/DNA130%20User%20Manual%20final%20versionpdf.pdf
 

SECTION: Probe synthesis
10.6. Add 6.5 ul of the "probe set" (ca. 500 ng) to each reaction. Vortex and pulse centrifuge. 
SECTION: Probe synthesis
10.7. Incubate in a thermalcycler at:

37 deg C for 16 hr
In the last 30 min, add 2 ul of TURBO DNase (2 U/ul, or 4 U for up to 10 ug). Pipette up and down to mix. 

SECTION: Probe synthesis
10.8. Combine replicate reactions in a 1.5 ml Lo-bind tube. Top up to 100 ul with RNase-free water.
SECTION: Probe synthesis
10.9. Cleanup using an RNeasy Mini Kit

 


Follow the manufacturer's instructions to purify the probes using an RNeasy Mini Kit: 

http://www.bea.ki.se/documents/EN-RNeasy%20handbook.pdf

Elute in 60 ul RNase-free water. 
SECTION: Probe synthesis
10.10. Add 2.5 ul of SUPERase-IN RNAse Inhibitor (20 U/ul) to the eluate. Flick to mix and pulse centrifuge. 
SECTION: Probe synthesis
10.11. Qubit

Measure the concentration of the probes using the Qubit RNA kit following the manufacturer's instructions. 

https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017209_Qubit_4_Fluorometer_UG.pdf
 

SECTION: Probe synthesis
10.12. Fragment analyse

Examine the fragment length distribution of the probes using a fragment analyser such as the LabChip GXII RNA kit (or equivalent fragment analyser) following the manufacturer's instructions:

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

SECTION: Probe synthesis
10.13. Dilute the RNA probes to 200 ng/ul with RNAse-free water. Aliquot into 6 ul batches in 0.5 ml Lo-bind Safelock tubes and store at -80 deg C. 
SECTION: Hybridisation
11. Label tubes. 
 
 

SECTION: Hybridisation
12. Ensure all primer stocks are pepared in the correct buffer and that enough aliquots of the working concentrations are diluted out. Vortex and pulse centrifuge. 
SECTION: Hybridisation
13. Thaw reagents. Flick all reagents to mix and pulse centrifuge where possible. 
 

SECTION: Hybridisation
14. Combine the following "BLOCKS" master mix in a 1.5 ml Lo-bind Safelock tube. Vortex and pulse centrifuge. 
 
 

SECTION: Hybridisation
16. Combine the following "HYBS" master mix in a 1.5 ml Lo-bind Safelock tube. Vortex and pulse centrifuge. 
 
 
 

SECTION: Hybridisation
15. Aliquot 3 ul of the BLOCKS master mix into 0.2 ml Lo-bind tubes, one for each capture reaction. 
SECTION: Hybridisation
17. Aliquot 14.5 ul of the HYBS master mix into 0.2 ml Lo-bind tubes, one for each capture reaction. 
SECTION: Hybridisation
18. Bring all the tubes to the post-PCR space. 
SECTION: Hybridisation
19. Pre-program the thermal cycler:

    95 deg C for 5 min
    60 deg C for 5 min
    60 deg C for hold
 

SECTION: Hybridisation
20. Thaw a 6-ul aliquot of the probes from Step 10.13 on ice. 
SECTION: Hybridisation
21. Add 5.5 ul of baits (ca. 500-1000 ng at 100-200 ng/ul) to each HYBS reaction. Flick to mix and pulse centrifuge. Place on ice.
SECTION: Hybridisation
22. Add 7 ul (100-500 ng) of shotgun library to the corresponding BLOCKS tube. Flick to mix and pulse centrifuge. 
 

SECTION: Hybridisation
23. Transfer the BLOCKS tubes to a thermalcycler and start the program. Allow it to proceed through Step 1 (i.e., 95 deg C for 5 min).

SECTION: Hybridisation
24. When the thermalcycler reaches Step 2, transfer the HYBS tubes to the thermalcyler. Allow it to proceed through Step 2 (i.e., 60 deg C for 5 min). 
SECTION: Hybridisation
25. When the thermalcycler reaches Step 3, transfer 18 ul from the HYBS tubes to the corresponding BLOCKS tubes. Pipette to mix. Discard the HYBS tubes. 
SECTION: Hybridisation
26. Add 15 ul of mineral oil to the top of each reaction. 
SECTION: Hybridisation
27. Allow the thermalcycler to proceed through Step 3 for 42 hr (i.e., 60 deg C for 42 hr). 
SECTION: Enrichment
28. Label tubes. 
 
 
 

SECTION: Enrichment
29. Prepare all necessary buffers and UV decontaminate. 
 

SECTION: Enrichment
30. Aliquot reagents for to take to the post-PCR space. 
 

SECTION: Library amplification
53. Thaw reagents on ice. 
SECTION: Library amplification
54. Ensure that all primer stocks are prepared in the correct buffer and that enough aliquots of the working concentrations are diluted out. Vortex and pulse centrifuge. 
 

SECTION: Enrichment
32. Set a heat block to 95 deg C. 
SECTION: Enrichment
33. Pellet the MyOne C1 Streptavidin beads for 2 min with the magnetic rack. Discard the supernatant. 
SECTION: Enrichment
31. Set a water bath or thermalshaker to 55 deg C. Warm Wash buffer 2.2 to 55 deg C for 45 min. 
SECTION: Enrichment
34. Add 200 ul * # reactions of Binding buffer to the beads. Vortex and pulse centrifuge. 
SECTION: Enrichment
35. Pellet the beads with the magnetic rack and discard the supernatant. 
SECTION: Enrichment
36. Repeat Step 34-35 two more times for a total of 3 washes. 
SECTION: Enrichment
37. Resuspend the beads in 70 ul * # reactions of Binding buffer. 
SECTION: Enrichment
38. Aliquot 70 ul of beads into a 1.5 ml Lo-bind Eppendorf tube (1 per reaction).
SECTION: Enrichment
39. Warm the bead aliquots to 55 deg C in the thermoshaker/water bath for 2 minutes. 
SECTION: Enrichment
40. Transfer the hybridised libraries at 60 deg C to the bead aliquots. Pipette to mix. 
SECTION: Enrichment
41. Incubate the hybridised libraries and beads in the thermoshaker/waterbath for 30 min at 55 deg C. Agitate every 5 minutes by flicking or gently continuously shake. 
SECTION: Enrichment
42. Pulse centrifuge. Pellet the beads with the magnetic rack. Discard the supernatant. 
SECTION: Enrichment
43. Add 500 ul heated Wash Buffer 2.2 to the beads. Vortex and pulse centrifuge. 
SECTION: Enrichment
44. Incubate 10 min at 55 deg C in the thermoshaker/water bath. Agitate every 2 min by flicking. 
SECTION: Enrichment
45. Pulse centrifuge. Pellet the beads with the magnetic rack. Discard the supernatant. 
SECTION: Enrichment
46. Repeat the wash steps above (Steps 43-45) two more times for a total of 3 washes. 
SECTION: Enrichment
47. Add 30 ul of 10 mM Tris-HCl/0.05% Tween-20 to the washed beads. Resuspend by pipetting. 
SECTION: Enrichment
48. Incubate at 95 deg C in a heat block for 5 min.
SECTION: Enrichment
49. Pellet the beads with a magnetic rack and transfer the supernatant to a clean 1.5 ml tube. 




SECTION: Enrichment
50. At this point you can treat with RNAseA to remove any RNA or put RNAse A in the PCR but it is not necessary as the RNA will not amplify. 
SECTION: Library amplification
55. Make up the following master mix in a 1.5 ml Lo-bind tube.
 
 
 

 
SECTION: Library amplification
56. Add 22.5 ul of master mix to each PCR tube following the schematic below. Pulse centrifuge the tubes. 

 

SECTION: Library amplification
57. Make up the following master mix in a 1.5 ml Lo-bind tube.


        ABCDEF1ReagentV2C1C2V1x _____ rxn2Ultrapure water25 ulnana10.93BSA25 ul10 mg /ml0.4 mg/ml1 ul4ABI Gold PCR Buffer25 ul10 X1 X2.5 ul5MgCl225 ul25 mM2.5 mM2.5 ul6dNTPs25 ul25 mM0.25 mM0.25 ul7ABI Taq Gold DNA polymerase25 ul5 U/ul0.05 U/ul0.25 ul8SYBR Green25 ul5 X0.12 X0.6 ul9P525 ul10 uM0.4 uM1 ul10P725 ul10 uM0.4 uM1 ul

SECTION: Library amplification
52. Label tubes.
SECTION: Enrichment
51. Dilute the captured libraries 1 in 10 in Ultrapure water (i.e., 1 ul in 9 ul Ultrapure water). Vortex and pulse centrifuge.
SECTION: Library amplification
58. Add 20 ul of master mix to each PCR tube following the schematic below. Pulse centrifuge the tubes. 

 

 
 
SECTION: Library amplification
59. Pulse centrifuge the 8-well qPCR strip tubes containing the master mix. Bring to the post-PCR space.
SECTION: Library amplification
60. To the end-point PCR, add 2.5 ul of both neat and 1in10 captured library to the corresponding tubes. Add 2.5 ul nuclease free water to the remaining wells as PCR no-template controls. Vortex and pulse centrifuge. 
SECTION: Library amplification
61. Place tubes in the thermal cycler and run the following PCR 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: Library amplification
62. When the PCR is finished, determine the optimal number of cycles to give in the final PCR to ensure libraries are not over-amplified. i.e., stop the PCR during the linear phase of amplification.
 

SECTION: Library amplification
64. To the second PCR add 5 ul of the neat library (as long as amplification was NOT inhibited in the first PCR; if amplification efficiency was poor, amplify a dilution and do more replicates). Perform enough replicates to amplify the entire captured library.  

SECTION: Library amplification
63. Agarose gel electrophoresis

Run 10 ul of PCR product from the first PCR on a 2% agarose gel electrophoresis. 
 

SECTION: Library amplification
65. Perform Step 61 above, but stop the PCR during the linear phase of amplification as determined by the end-point PCR above. 
 

SECTION: Library amplification
66. Combine replicates in a clean 1.5 mL Lo-bind Safelcok tube. Vortex and pulse centrifuge. 
SECTION: Library amplification
67. Purify

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 40 ul of Ultrapure water. 
SECTION: Size select enriched libraries
68. Run each enriched library 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 enriched libraries
69. Purify

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: Pool enriched libraries
70. Qubit

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: Pool enriched libraries
72. Fragment analyse

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 enriched libraries
71. Dilute the libraries to 5 ng/ul in Ultrapure water in a total volume of 5-10 ul. 
SECTION: Pool enriched libraries
73. Pool enriched libraries in equimolar concentrations.
SECTION: Quantitate the final sequencing library
74. 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 sequencing library
75. Qubit

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 sequencing library
76. Fragment analyse

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 sequencing library
77. 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: Sequence
78. Dilute the library to between 2-4 nM in Ultrapure water. 
 

SECTION: Sequence
79. Follow the manufacturer's instructions to perform the sequencnig run on your Illumina platform of choice.