DSBEST v2.0 (Caroe et al. 2018)
Alicia Grealy
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
This bench protocol is based on the work of Caroe et al. (2018), for preparing shotgun libraries from double-stranded DNA, typically for ancient and degraded DNA. Please cite Caroe et al. (2018) if you use this bench protocol!
Before start
I have begun including a double-stranded positive control oligo (dsCL104) in the library preparation; this is a double-stranded form of the single-stranded oligo described in Gansauge and Meyer (2013). Always include no-template controls and any extraction controls.
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.
Attachments
Steps
Preparation
"Suit up" in this order: hair net, nitrile gloves, facemask, coveralls, gumboots, booties, second pair of gloves.
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.
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.
Label tubes.
| A | B | C |
|---|---|---|
| Tube | Qty | For ... |
| 1.5 ml Safelock Tube | 10 | 5X SYBR |
| 0.5 ml Safelock Tube | 8 | 25 mM dNTPs |
| 1.5 ml Safelock Tube | 1 | 0.1 uM CL104_duplex |
| 1.5 ml Safelock Tube | 1 | 0.1 uM CL105_duplex 1/500 |
| 1.5 ml Safelock Tube | 1 | 10 uM IS7 |
| 1.5 ml Safelock Tube | 1 | 10 uM IS8 |
| 1.5 ml Safelock Tube | 1 | 10 uM CL107 |
| 1.5 ml Safelock Tube | 1 | 10 uM CL108 |
| 1.5 ml Safelock Tube | 10 | CL105_106 STD dilution series 10^11 - 10^2 |
| 15 ml Falcon Tube | 1 | TE Buffer |
| 15 ml Falcon Tube | 1 | TET buffer |
| 1.5 ml Safelock Tube | 1 | Oligo hybridisation buffer |
| 1.5 ml Safelock Tube | 1 | Reaction enhancer |
| 15 ml Falcon Tube | 1 | EBT buffer |
| 0.2 ml Lo-bind PCR tube | 2 | Adapters (P5_DS1, P7_DS2) |
| 0.5 ml Lo-bind PCR tube | 1 | Adapters mix (DS_adapter_mix) |
| 0.5 ml Lo-bind PCR tube | 1 | Adapters mix dilution |
| 0.2 ml Lo-bind PCR Tube | # of samples + 2 | Reaction tubes |
| 1.5 ml Lo-bind Tube | 3 | Step 19, Step 24, Step 27 master mixes |
| 1.5 ml Lo-bind Tube | # of samples + 2 | Combining library with PB buffer |
| 1.5 ml Lo-bind Tube | # of samples + 2 | Elution of library from spin column |
| QIAGEN MinElute PCR Purification Spin Columns | # of samples + 2 | Purification of library |
| 0.5 ml Lo-bind Tube | # of samples + 2 | Final library |
| 0.5 ml Lo-bind Tube | # of samples + 2 | 1/20 dilution of library |
| 1.5 ml Safelock Tube | 2 | Assay A and B master mixes |
| 8-strip optical qPCR Tubes | (((# of samples + 2)*2)+26)/8 | Assay A and B |
Prepare all necessary buffers and UV decontaminate where appropriate.
| A | B | C | D |
|---|---|---|---|
| Buffer | Reagent | Volume to add | Final concentration in solution |
| Oligo hybridisation buffer 10X | 5 M NaCl | 100 ul | 500 mM |
| 1 M Tris-HCl | 10 ul | 10 mM | |
| 0.5 M EDTA | 2 ul | 1 mM | |
| Ultrapure water | 888 ul | na | |
| Reaction enhancer | 50% PEG-4000 | 500 ul | 25% |
| 10 mg/ml BSA | 200 ul | 2 mg/ml | |
| 5 M NaCl | 80 ul | 400 mM | |
| Ultrapure water | 220 ul | na | |
| TE Buffer | Ultrapure water | 9.88 ml | na |
| (Exp. 1 year) | 1 M Tris-HCl | 100 ul | 0.01 M |
| 0.5 M EDTA | 20 ul | 0.001 M | |
| EBT | 1 M Tris-HCl | 100 ul | 0.01 M |
| 100% Tween 20 | 5 ul | 0.05% | |
| Ultrapure water | 9.895 ml | na | |
| TET buffer | 1 M Tris-HCl | 500 ul | 0.01 M |
| (Exp. 1 year) | 0.5 M EDTA | 100 ul | 0.001 M |
| 100% Tween 20 | 25 ul | 0.05% | |
| Ultrapure water | 49.375 ml | na | |
| 5X SYBR | 10,000X SYBR | 2.5 ul | 5X |
| DMSO | 997.5 ul | na | |
| DMSO | 4 ml | na | |
| 25 mM dNTPs | 100 mM dATP | 100 ul | 25 mM |
| 100 mM dTTP | 100 ul | 25 mM | |
| 100 mM dCTP | 100 ul | 25 mM | |
| 100 mM dGTP | 100 ul | 25 mM |
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!
| A | B | C |
|---|---|---|
| Working stock | Reagent | Volume to add |
| 10 uM CL104_duplex | 100 uM CL104 | 50 ul |
| TET buffer | 450 ul | |
| 0.1 uM CL104_duplex | 10 uM CL104 | 5 ul |
| TET buffer | 495 ul | |
| 0.1 uM CL104_duplex 1/500 | 0.1 uM CL104 | 1 ul |
| (i.e., 0.0002 uM) | TET buffer | 499 ul |
| 10 uM IS7 | 100 uM IS7 | 50 ul |
| Ultrapure water | 450 ul | |
| 10 uM IS8 | 100 uM IS8 | 50 ul |
| Ultrapure water | 450 ul | |
| 10 uM CL107 | 100 uM CL107 | 50 ul |
| Ultrapure water | 450 ul | |
| 10 uM CL108 | 100 uM CL108 | 50 ul |
| Ultrapure water | 450 ul | |
| 10 uM CL105_106_STD | 100 uM CL105_106_STD | 50 ul |
| TET buffer | 450 ul | |
| 10^11 copies CL105_106_STD | 10 uM CL105_106_STD | 10 ul |
| TET buffer | 592.25 ul | |
| 10^10 copies CL105_106_STD | 10^11 copies CL105_106_STD | 50 ul |
| TET buffer | 450 ul | |
| 10^9 copies CL105_106_STD | 10^10 copies CL105_106_STD | 50 ul |
| TET buffer | 450 ul | |
| 10^8 copies CL105_106_STD | 10^9 copies CL105_106_STD | 50 ul |
| TET buffer | 450 ul | |
| 10^7copies CL105_106_STD | 10^8 copies CL105_106_STD | 50 ul |
| TET buffer | 450 ul | |
| 10^6 copies CL105_106_STD | 10^7copies CL105_106_STD | 50 ul |
| TET buffer | 450 ul | |
| 10^5 copies CL105_106_STD | 10^6 copies CL105_106_STD | 50 ul |
| TET buffer | 450 ul | |
| 10^4 copies CL105_106_STD | 10^5 copies CL105_106_STD | 50 ul |
| TET buffer | 450 ul | |
| 10^3 copies CL105_106_STD | 10^4 copies CL105_106_STD | 50 ul |
| TET buffer | 450 ul | |
| 10^2 copies CL105_106_STD | 10^3 copies CL105_106_STD | 50 ul |
| TET buffer | 450 ul |
Pre-program the thermal cycler.
Prepare adapters
In a 0.2 ml Lo-bind PCR tube ("P5_DS1"), combine:
40 ul of 500 uM IS1
40 ul of 500 uM IS3
10 ul Ultrapure water
10 ul 10X Oligo Hybridisation Buffer
Vortex and pulse centrifuge.
In a new 0.2 ml Lo-bind PCR tube ("P7_DS2"), combine:
40 ul of 500 uM IS2
40 ul of 500 uM IS3
10 ul Ultrapure water
10 ul 10X Oligo Hybridisation Buffer
Vortex and pulse centrifuge.
Incubate both "P5_DS1" and "P7_DS2" in a thermal cycler:
95 deg C for 10 sec
Ramp down to 12 deg C at a rate of 0.1 deg C/sec
Combine both "P5_DS1" and "P7_DS2" together in a 0.5 ml Lo-bind Safelock tube to give 200 ul of 100 uM "DS_adapter_mix".
Determine the DNA input amount and concentration of adapters required
Measure the concentration of your DNA extract on the Qubit following the manufacturer's instructions.
https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017209_Qubit_4_Fluorometer_UG.pdf
If possible, run the DNA on a fragment analyser or gel electrophoresis to determine the fragment length distribution.
https://www.perkinelmer.com/Content/LST_Software_Downloads/LabChip_GX_User_Manual.pdf
2% Agarose Gel Electrophoresis
Calculate the molarity of the dsDNA ends . Use the calculator at:
https://nebiocalculator.neb.com/#!/dsdnaends
Calculate the pmol of adapters needed. This is approximately 10X the molarity of the dsDNA ends. Round up to the nearest 10 pmol.
This number is equal to the concentration of adapters needed in uM.
Dilute the adapters to the desired working concentration in EBT buffer.
End Repair
Combine the following in a 1.5 ml Lo-bind Safelock tube. Vortex and pulse centrifuge.
| A | B | C | D | E | F |
|---|---|---|---|---|---|
| Reagent | V2 (reaction volume) | C1 (stock concentration) | C2 (concentration in reaction) | V1 (volume to add) | x _______ rxn |
| T4 DNA ligase buffer w/ 10 mM ATP (NEB) | 40 ul | 10 X | 1 X | 4 ul | |
| T4 DNA Polymerase | 40 ul | 3 U/ul | 0.03 U/ul | 0.4 ul | |
| T4 PNK | 40 ul | 10 U/ul | 0.25 U/ul | 1 ul | |
| Reaction enhancer | 40 ul | na | na | 2.2 ul | |
| dNTPs | 40 ul | 25 mM | 0.25 mM | 0.4 ul |
Aliquot 8 ul into a 0.2 ml Lo-bind PCR tube for each reaction.
To make the total reaction volume up to 40 ul, add:
Up to 32 ul DNA to each sample reaction. Make up the remainder with Ultrapure water. Typically input 3x10^8 - 3x10^11 double-stranded molecules; 13 pg-14 ng of ca. 40 bp DNA--this is typically 20% of the extract.
1 ul of 0.1 uM CL104_duplex positive control oligo to the positive conrol reaction + 31 ul Ultrapure water. We are inputing 3.01x10^10 molecules of single-stranded CL104 into the library preparation.
32 ul of Ultrapure water to the no-template control reaction.
Vortex and pulse centrifuge.
Incubate in a thermal cycler at:
20 deg C for 30 min
65 deg C for 30 min
4 deg C hold
Adapter ligation
Add 1 ul of the DS_adapter_mix DILUTION (e.g., 10 uM) to the finished end-repair reaction above. Vortex and pulse centrifuge.
Combine the following in a 0.5 ml Lo-bind tube. Vortex and pulse centrifuge.
| A | B | C | D | E | F |
|---|---|---|---|---|---|
| Reagent | V2 (reaction volume) | C1 (stock concentration) | C2 (concentration in reaction) | V1 (volume to add) | x _______ rxn |
| T4 DNA ligase buffer w/ 10 mM ATP (NEB) | 50 ul | 10 X | 0.2 X | 1 ul | |
| PEG-4000 | 50 ul | 50% | 6% | 6 ul | |
| T4 DNA ligase | 50 ul | 400 U/ul | 8 U/ul | 1 ul | |
| Ultrapure water | 50 ul | na | na | 1 ul |
Add 9 ul to each reaction. Vortex and pulse centrifuge.
Incubate in a thermal cycler at:
20 deg C for 30 min
65 deg C for 10 min
4 deg C hold
Fill in
Combine the following in a 0.5 ml Lo-bind tube. Vortex and pulse centrifuge.
| A | B | C | D | E | F |
|---|---|---|---|---|---|
| Reagent | V2 (reaction volume) | C1 (stock concentration) | C2 (concentration in reaction) | V1 (volume to add) | x _______ rxn |
| Isothermal amplification buffer | 60 ul | 10 X | 0.33 X | 2 ul | |
| dNTPs | 60 ul | 25 mM | 0.33 mM | 0.8 ul | |
| Bst 2.0 Warmstart DNA polymerase | 60 ul | 8 U/ul | 0.213 U/ul | 1.6 ul | |
| Ultrapure water | 60 ul | na | na | 5.6 ul |
Add 10 ul of the above mix to each reaction. Vortex and pulse centrifuge.
Incubate in a thermal cycler at:
65 deg C for 15 min
80 deg C for 15 min
4 deg C hold
Purify library
Purify the libraries using a QIAGEN MinElute PCR Purification kit.
https://www.qiagen.com/au/resources/resourcedetail?id=e24a0a3a-e9cb-4180-a6b4-202c527b924c&lang=en
Briefly...
Combine 300 ul of PB Buffer with each reaction in a 1.5 ml Lo-Bind tube. Vortex and pulse centrifuge.
Transfer the mixture to a MinElute Silica Spin Column (purple) placed inside a collection tube. Centrifuge for 1 min at 13,000 rpm in a bench-top centrifuge. Discard the flow-through.
Add 700 ul of PE Buffer to the column. Centrifuge for 1 min at 13,000 rpm in a bench-top centrifuge. Discard the flow-through.
Repeat Step 29.
Centrifuge one more time (dry) for 1 min at 13,000 rpm in a bench-top centrifuge. Place the column in a clean 1.5 ml Lo-bind tube with the lid cut off.
Add 25 ul of EB buffer to the column. Incubate 5 min at room temperature.
Centrifuge 1 min at 13,000 rpm. Add the eluate back through the column to increase yield. Incubate for 5 min at room temperature. Centrifuge 1 min at 13,000 rpm.
Transfer the eluate to a clean 0.5 ml Lo-bind tube.
Dilute the library 1 in 20 in Ultrapure water (i.e., add 1 ul of the library to 19 ul of Ultrapure water). Vortex and pulse centrifuge.
Libraries can be stored at -20 deg C until amplification. For long-term storage, store at -80 deg C.
Quant the library
Make up the following master mix in a 1.5 ml Lo-bind tube. Vortex and pulse centrifuge.
| A | B | C | D | E | F |
|---|---|---|---|---|---|
| Reagent | V2 | C1 | C2 | V1 | x _______ rxn |
| Ultrapure water | 25 ul | na | na | 15.9 ul | |
| BSA | 25 ul | 10 mg /ml | 0.4 mg/ml | 1 ul | |
| ABI Gold PCR Buffer | 25 ul | 10 X | 1 X | 2.5 ul | |
| MgCl2 | 25 ul | 25 mM | 2.5 mM | 2.5 ul | |
| dNTPs | 25 ul | 25 mM | 0.25 mM | 0.25 ul | |
| ABI Taq Gold DNA polymerase | 25 ul | 5 U/ul | 0.05 U/ul | 0.25 ul | |
| SYBR Green | 25 ul | 5 X | 0.12 X | 0.6 ul | |
| IS7 | 25 ul | 10 uM | 0.2 uM | 0.5 ul | |
| IS8 | 25 ul | 10 uM | 0.2 uM | 0.5 ul |
Assay A master mix
Make up the following master mix in a 1.5 ml Lo-bind tube. Vortex and pulse centrifuge.
| A | B | C | D | E | F |
|---|---|---|---|---|---|
| Reagent | V2 | C1 | C2 | V1 | x 16_ rxn |
| Ultrapure water | 25 ul | na | na | 15.9 ul | 254.4 ul |
| BSA | 25 ul | 10 mg /ml | 0.4 mg/ml | 1 ul | 16 ul |
| ABI Gold PCR Buffer | 25 ul | 10 X | 1 X | 2.5 ul | 40 ul |
| MgCl2 | 25 ul | 25 mM | 2.5 mM | 2.5 ul | 40 ul |
| dNTPs | 25 ul | 25 mM | 0.25 mM | 0.25 ul | 4 ul |
| ABI Taq Gold DNA polymerase | 25 ul | 5 U/ul | 0.05 U/ul | 0.25 ul | 4 ul |
| SYBR Green | 25 ul | 5 X | 0.12 X | 0.6 ul | 9.6 ul |
| CL107 | 25 ul | 10 uM | 0.2 uM | 0.5 ul | 8 ul |
| CL108 | 25 ul | 10 uM | 0.2 uM | 0.5 ul | 8 ul |
Assay B master mix
Add 24 ul of master mix to the corresponding PCR tubes. Pulse centrifuge the tubes.
Add 1 ul of DNA sample to the corresponding PCR tubes according to the scheme below. Pulse centrifuge the tubes.
| A | B | C | D |
|---|---|---|---|
| PCR NTC | CL105_106_STD 10^3 | dsLib001 Neat | dsLib005 Neat |
| PCR NTC | CL105_106_STD 10^3 | dsLib001 1in20 | dsLib005 1in20 |
| CL105_106_STD 10^6 | CL105_106_STD 10^2 | dsLib002 Neat | ...etc. |
| CL105_106_STD 10^6 | CL105_106_STD 10^2 | dsLib002 1in20 | |
| CL105_106_STD 10^5 | dsCL104 +VE Neat | dsLib003 Neat | |
| CL105_106_STD 10^5 | dsCL104 +VE 1in20 | dsLib003 1in20 | |
| CL105_106_STD 10^4 | dsNTC -VE Neat | dsLib004 Neat | |
| CL105_106_STD 10^4 | dsNTC -VE 1in20 | dsLib004 1in20 |
Assay A Plate set-up
| A | B |
|---|---|
| 0.1 uM CL104_duplex 1/500 | CL105_106_STD 10^4 |
| 0.1 uM CL104_duplex 1/500 | CL105_106_STD 10^4 |
| PCR NTC | CL105_106_STD 10^3 |
| PCR NTC | CL105_106_STD 10^3 |
| CL105_106_STD 10^6 | CL105_106_STD 10^2 |
| CL105_106_STD 10^6 | CL105_106_STD 10^2 |
| CL105_106_STD 10^5 | |
| CL105_106_STD 10^5 |
Assay B Plate set-up
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
Electrophorese 10 ul of the PCR product from the libraries (not standards) and controls on a 2% agarose gel.
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.
Index/amplify the library
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.
| A | B | C | D | E | F |
|---|---|---|---|---|---|
| Reagent | V2 | C1 | C2 | V1 | x _____ rxn |
| Ultrapure water | 25 ul | na | na | 10.9 | |
| BSA | 25 ul | 10 mg /ml | 0.4 mg/ml | 1 ul | |
| ABI Gold PCR Buffer | 25 ul | 10 X | 1 X | 2.5 ul | |
| MgCl2 | 25 ul | 25 mM | 2.5 mM | 2.5 ul | |
| dNTPs | 25 ul | 25 mM | 0.25 mM | 0.25 ul | |
| ABI Taq Gold DNA polymerase | 25 ul | 5 U/ul | 0.05 U/ul | 0.25 ul | |
| SYBR Green | 25 ul | 5 X | 0.12 X | 0.6 ul | |
| P5_indexing_primer | 25 ul | 10 uM | 0.2 uM | 0.5 ul | Don't add to master mix |
| P7_indexing_primer | 25 ul | 10 uM | 0.2 uM | 0.5 ul | Don't add to master mix |
Add 19 ul of master mix to the corresponding PCR tubes. Pulse centrifuge the tubes.
Add 0.5 ul of the corresponding forward indexing primer to the appropriate reaction tube. Pulse centrifuge the tubes.
Add 0.5 ul of the corresponding reverse indexing primer to the appropriate reaction tube. Pulse centrifuge the tubes.
Add 5 ul of DNA sample to the corresponding reaction tubes according to the scheme below. Pulse centrifuge the tubes.
e.g.,
| A | B | C | D | E | F |
|---|---|---|---|---|---|
| dsLib001 | dsLib003 | dsLib005 | |||
| dsLib001 | dsLib003 | dsLib005 | |||
| dsLib001 | dsLib003 | dsLib005 | |||
| dsLib001 | dsLib003 | dsLib005 | |||
| dsLib002 | dsLib004 | ...etc. | |||
| dsLib002 | dsLib004 | ||||
| dsLib002 | dsLib004 | ||||
| dsLib002 | dsLib004 |
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
Purify the libraries
Pulse centrifuge the PCR tubes. Combine replicate PCR reactions into a 1.5 ml Lo-bine Safelock tube. Vortex and pulse centrifuge.
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.
Quantitate the libraries
Dilute the libraries 1 in 10 in Ultrapure water (i.e., 1 ul library in 9 ul Ultrapure water).
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
Pool libraries
Pool libraries in equimolar concentrations such that the total amount of DNA per library does not exceed 500-1000 ng.
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.
Alternatively, concentrate the libraries using a SpeedyVac system, following the manufacturer's instructions.
Size select and purify
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
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.
Quantitate the final library
Dilute the libraries 1/2, 1/5, 1/10 in Ultrapure water (i.e., create a serial dilution in 10 ul volume).
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
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
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.
Sequencing
Dilute the library to between 2-4 nM in Ultrapure water.
Follow the manufacturer's instructions to perform the sequencnig run on your platform of choice.
