An overview of the whole workflow is shown on Figure 1.
NEBNext Enzymatic Methyl-Seq Protocol Standard Insert Library (370-420bp2)
👉 All reagents thaw on ice unless specific recommendations, then pulse-vortex 2sec to mix, and followed by pulse-spin
1. Mechanical fragmentation (Figure 1)
1.1. Genomic DNA shearing (Figure 1)
▢ Dilute genomic DNA (50ng* or 100ng**2, fluorometric assay) to 50µl with EB buffer
🔖 *50ng per semence extraction and **100ng per blood extraction
▢ Transfer the diluted DNA in a tube appropriated to an ultrasonicator instrument
▢ Fragment the genomic DNA using Covaris ultrasonication parameters 2
Repeat : 20 iterations
Process : dithering
Treatment :
Duration : 10sec
Peak Power : 210W
Duty Factor : 25%
Cycles Per Burst : 50
Average Power : 52.5W
🔴 Possible stop point post-fragmentation but avoid the DNA freeze thaws
1.2. Optional QC fragmentation (Figure 1)
1.2.1. Quantification is performed on a Qubit 3.0 fluorometer with DS DNA High Sensitivity Quantitation Assay kit according manufacturer’s recommendations (Agilent, ThermoFisher Scientific)
1.2.2. Validation is performed on a 2100 Bioanalyzer with DNA 7500 kit according manufacturer’s recommendations (Agilent Technologies)
📝 Average size distribution should be approximately 240-290bp2
2. Library preparation (Figure 1)
2.1. End repair/dA-tailing (Figure 1)
▢ Transfer 50µl of the sheared DNA in a 0.2ml tube appropriated to a thermocycler instrument
▢ On ice, add the following components to the 0.2ml tube of sheared DNA :
▢ NEBNext Ultra II End prep Reaction Buffer (green) : 7µl
▢ NEBNext Ultra II End prep Enzyme Mix (green) : 3µl
Total volume : 60µl
▢ Set a 200µl pipette to 50µl and then gently pipette the entire volume up and down at least 10 times to mix thoroughly
▢ Perform a quick spin to collect all liquid from the sides of the 0.2ml tube
📝 The presence of small amount of bubbles will not interfere with performance
▢ Place the 0.2ml tube in a thermocycler and run the following program :
Temperature Time
20°C 30min
65°C 30min
4°C hold
2.2. Methylated adaptor ligation (Figure 1)
▢ On ice, add the following components to the 0.2ml tube of repaired DNA :
▢ NEBNext® EM-seq™ Adaptor (red) : 2.5µl
Total volume : 62.5µl
▢ Set a 200µl pipette to 50µl and then gently pipette the entire volume up and down at least 10 times to mix thoroughly
▢ Perform a quick spin to collect all liquid from the sides of the 0.2ml tube
▢ On ice, add the following components to the 0.2ml tube of repaired DNA :
▢ NEBNext® Ligation Enhancer (red) : 1µl
▢ NEBNext® Ultra™ II Ligation Master Mix (red) : 30µl
Total volume : 93.5µl
📝 The Ligation Master Mix is viscous
👉 For multiple reactions, a master mix of above reaction components can be prepare before addition to the mix sample/adaptor
▢ Set a 200µl pipette to 80µl and then gently pipette the entire volume up and down at least 10 times to mix thoroughly
▢ Perform a quick spin to collect all liquid from the sides of the 0.2ml tube
📝 The presence of small amount of bubbles will not interfere with performance
▢ Incubate at 20°C for 15min in a thermocycler with the heated lid turned off
🔴 Safe stopping point : Sample can be stored overnight at -20°C
2.3. Clean-up of adaptor ligated DNA (Figure 1)
▢ Equilibrate NEBNext® Sample Purification Beads to room temperature (30min)
▢ Vortex NEBNext® Sample Purification Beads until homogenization
▢ Add 110µl (1.18X) of homogenized beads to each sample
▢ Mix well by vortexing (until homogenization)
▢ Incubate 5min at room temperature
▢ Place 0.2ml tube against an appropriate magnetic stand for 5min (or when the solution is clear) to separate the beads from the supernatant
▢ Carefully remove and discard supernatant
📝 Do not discard beads pellet that contain DNA targets
▢ Add 200µl of 80% freshly prepared 80% ethanol to gently wash the beads pellet
📝 Do not disturb the bead pellet
▢ Incubate 1min
▢ Carefully remove and discard ethanol
📝 Do not discard beads that contain DNA targets
▢ Add 200µl of 80% freshly prepared 80% ethanol to gently wash the beads pellet
📝 Do not disturb the beads pellet
▢ Incubate 1min
▢ Carefully remove and discard ethanol
📝 Do not discard beads that contain DNA targets
📝 Be sure to remove all visible liquid using a 10µl pipette tip
▢ Air-dry the beads pellet for 2min (or until the beads pellet is dry)
👉 Do not over-dry the beads pellet to not reduce DNA recovery. The beads must still remain dark brown and glossy looking ; the beads must not turn lighter and start to crack
▢ Remove the 0.2ml tube from the magnetic stand
▢ Add 30µl of Elution Buffer (white) to the beads pellet
▢ Mix well by pipetting up and down 10 times (until homogenization)
▢ If necessary, quickly spin the sample to collect the liquid from the sides of the 0.2ml tube
▢ Incubate 2min at room temperature
▢ Place the 0.2ml tube against an appropriate magnetic stand for 3min (until the solution is clear) to separate the beads from the supernatant
▢ Transfer 28µl of the clear supernatant containing the adaptor ligated DNA to a new 0.2ml PCR tube
📝 Make sure not to disturb the beads pellet
🔴 Safe stopping point : Sample can be stored overnight at -20°C
2.4. Optional QC ligation (Figure 1)
2.4.1. Quantification is performed on a Qubit 3.0 fluorometer with DS DNA High Sensitivity Quantitation Assay kit according manufacturer’s recommendations (Agilent, ThermoFisher Scientific)
2.4.2. Validation is performed on a 2100 Bioanalyzer with DNA 7500 kit according manufacturer’s recommendations (Agilent Technologies)
📝 Average size distribution should be approximately 370-420bp2
3. Enzymatic conversion (Figure 1)
3.1. Oxidation of 5mC and 5hmC (Figure 1)
▢ Add the entire volume of TET2 Reaction Buffer (yellow) to TET2 Reaction Buffer Supplement powder (yellow)
▢ Mix well and mention the date
👉 Reconstituted TET2 Reaction Buffer should be stored at -20°C and discarded after 4 months
▢ On ice, add the following components to the 28µl adaptor ligated DNA :
▢ Reconstituted TET2 Reaction Buffer (yellow) 10µl
▢ Oxidation Supplement (yellow) 1µl
▢ DTT (yellow) 1µl
▢ Oxidation Enhancer (yellow) 1µl
▢ TET2 (yellow) 4µl
Volume total : 45µl
👉 For multiple reactions, a master mix of above reaction components can be prepared before addition to the sample
▢ Mix thoroughly by vortexing
▢ Centrifuge briefly
▢ Dilute the 500nM Fe (II) solution (yellow) by adding 1µl to 1249µl of nuclease-free water
⚠ Use the diluted Fe (II) solution immediately, discard after use
▢ Add 5µl of diluted Fe (II) solution to the 0.2ml tube to the initiated oxidation reaction
Volume total : 50µl
▢ Mix well by vortexing
▢ Centrifuge briefly
▢ Incubate at 37°C for 1hr in a thermocycler
▢ Transfer the sample on ice
▢ Add 1µl of Stop Reagent (yellow)
▢ Mix well by vortexing
▢ Centrifuge briefly
▢ Incubate at 37°C for 30min then at 4°C in a thermocycler
🔴 Possible stopping point : Sample can be stored overnight at either 4°C in the thermocycler or at -20°C in the freezer
3.2. Clean-up of oxidated DNA (Figure 1)
▢ Equilibrate NEBNext® Sample Purification Beads to room temperature (30min)
▢ Vortex NEBNext® Sample Purification Beads until homogenization
▢ Add 90µl (1.8X) of homogenized beads to each sample
▢ Mill well by vortexing (until homogenization)
▢ Incubate 5min at room temperature
▢ Place the 0.2ml tube against an appropriate magnetic stand for 5min (until the solution is clear) to separate the beads from the supernatant
▢ Carefully remove and discard supernatant
📝 Do not discard beads pellet that contain DNA targets
▢ Add 200µl of 80% freshly prepared ethanol to gently wash the beads pellet
📝 Do not disturb the beads pellet
▢ Incubate 1min
▢ Carefully remove and discard ethanol
📝 Do not discard beads that contain DNA targets
▢ Add 200µl of 80% freshly prepared ethanol to gently wash the beads pellet
📝 Do not disturb the beads pellet
▢ Incubate 1min
▢ Carefully remove and discard ethanol
📝 Do not discard beads that contain DNA targets
📝 Be sure to remove all visible liquid using a 10µl pipette tip
▢ Air-dry the beads pellet for 2min (until the beads pellet is dry)
👉 Do not over-dry the beads pellet to not reduce DNA recovery. The beads must still dark brown and glossy looking ; the beads must not turn lighter brown and start to crack
▢ Remove the 0.2ml tube from the magnetic stand
▢ Add 18µl of Elution Buffer (white) to the beads pellet
▢ If necessary, quickly spin the sample to collect the liquid from the sides of the 0.2ml tube
▢ Mix well by pipetting up and down 10 times (until homogenization)
▢ Incubate 2min at room temperature
▢ Place the 0.2ml tube against an appropriate magnetic stand for 3min (until the solution is clear) to separate the beads from the supernatant
▢ Transfer 16µl of the clear supernatant containing the converted DNA to a new 0.2ml PCR tube
📝 Make sure not to disturb the beads pellet
🔴 Safe stopping point : Sample can be stored overnight at -20°C
3.3. Optional QC oxidation (Figure 1)
3.4.1. Quantification is performed on a Qubit 3.0 fluorometer with DS DNA High Sensitivity Quantitation Assay kit according manufacturer’s recommendations (Agilent, ThermoFisher Scientific)
3.4.2. Validation is performed on a 2100 Bioanalyzer with DNA 7500 kit according manufacturer’s recommendations (Agilent Technologies)
📝 Average size distribution should be approximately 370-420bp2
3.4. Denaturation (⚠ under chemical fume cupboard) (Figure 1)
▢ Pre-heat a thermocycler to 85°C
▢ Add 4µl formamide to the 16µl of oxidized DNA
▢ Mix well by vortexing
▢ Centrifuge briefly
▢ Incubate at 85°C for 10min in the pre-heated thermocycler
▢ Immediately place on ice
3.5. Deamination of C to U (⚠ under chemical fume cupboard) (Figure 1)
▢ On ice, add immediately the following components to the 20µl denatured DNA :
▢ Nuclease water (orange) 68µl
▢ APOBEC Reaction Buffer (orange) 10µl
▢ BSA (orange) 1µl
▢ APOBEC (orange) 1µl
Volume total : 100µl
👉 For multiple reactions, a master mix of above reaction components can be prepared before addition to the denatured DNA
▢ Mix well by vortexing
▢ Centrifuge briefly
▢ Incubate at 37°C for 3hr then at 4°C in a thermocycler
🔴 Possible stopping point : Sample can be stored overnight at either 4°C in the thermocycler or at -20°C in the freezer
3.6. Clean-up of deaminated DNA (Figure 1) under chemical fume cupboard
▢ Equilibrate NEBNext® Sample Purification Beads to room temperature (30min)
▢ Vortex NEBNext® Sample Purification Beads until homogenization
▢ Add 100µl (1X) of homogenized beads to each sample
▢ Mill well by vortexing (until homogenization)
▢ Incubate 5min at room temperature
▢ Place the 0.2ml tube against an appropriate magnetic stand for 5min (until the solution is clear) to separate the beads from the supernatant
▢ Carefully remove and discard supernatant
📝 Do not discard beads pellet that contain DNA targets
▢ Add 200µl of 80% freshly prepared ethanol to gently wash the beads pellet
📝 Do not disturb the beads pellet
▢ Incubate 1min
▢ Carefully remove and discard ethanol
📝 Do not discard beads that contain DNA targets
▢ Add 200µl of 80% freshly prepared ethanol to gently wash the beads pellet
📝 Do not disturb the beads pellet
▢ Incubate 1min
▢ Carefully remove and discard ethanol
📝 Do not discard beads that contain DNA targets
👉 No more pipetting under fume cupboard at this step, so the beads don’t become too dry
📝 Be sure to remove all visible liquid using a 10µl pipette tip
▢ Air-dry the beads pellet for 1.5min (until the beads pellet is dry)
👉 Do not over-dry the beads pellet to not reduce DNA recovery. The beads must still dark brown and glossy looking ; the beads must not turn lighter brown and start to crack
👉 The beads behave differently during APOBEC clean-up, do not over-dry the beads as they become very difficult to resuspend
▢ Remove the 0.2ml tube from the magnetic stand
▢ Add 22µl of Elution Buffer (white) to the beads pellet
▢ Mix well by pipetting up and down 10 times (until homogenization)
▢ If necessary, quickly spin the sample to collect the liquid from the sides of the 0.2ml tube
▢ Incubate 2min at room temperature
▢ Place the 0.2ml tube against an appropriate magnetic stand for 3min (until the solution is clear) to separate the beads from the supernatant
▢ Transfer 20µl of the clear supernatant containing the deaminated DNA to a new PCR 0.2ml tube
📝 Make sure not to disturb the beads pellet
🔴 Safe stopping point : Sample can be stored overnight at -20°C
4. Indexing (Figure 1)
4.1. Indexation by PCR (Figure 1)
▢ On ice, add the following components to the 20µl deaminated DNA :
▢ EM-Seq Index Primer tube (10µM) 5µl
▢ NEBNext® Q5U® Master Mix (blue) 25µl
Total volume : 50µl
▢ Mix well by vortexing
▢ Centrifuge briefly
▢ Place the 0.2ml tube in a thermocycler and run the following program :
Cycles step Temperature Time Cycles2
Initial denaturation 98°C 30sec 1
Denaturation 98°C 10sec
Annealing 62°C 30sec 9* or 11**
Extension 65°C 60sec
Final extension 65°C 5min 1
Hold 4°C
🔖 **11 cycles for 50ng input and *9 cycles for 100ng input
🔴 Possible stopping point : Sample can be stored overnight at either 4°C in the thermocycler or at -20°C in the freezer
4.2. Clean-up indexed library (Figure 1)
▢ Vortex NEBNext® Sample Purification Beads until mixed well
▢ Add 45µl (0.9X2) of homogenized beads to each library
▢ Mill well by vortexing (until homogenization)
▢ Incubate 5min at room temperature
▢ Place the 0.2ml tube against an appropriate magnetic stand for 5min (until the solution is clear) to separate the beads from the supernatant
▢ Carefully remove and discard supernatant
📝 Do not discard beads that contain DNA targets
▢ Add 200µl of 80% freshly prepared ethanol to gently wash the beads pellet
📝 Do not disturb the beads pellet
▢ Incubate 1min
▢ Carefully remove and discard ethanol
📝 Do not discard beads that contain DNA targets
▢ Add 200µl of 80% freshly prepared ethanol to gently wash the beads pellet
📝 Do not disturb the beads pellet
▢ Incubate 1min
▢ Carefully remove and discard ethanol
📝 Do not discard beads that contain DNA targets
📝 Be sure to remove all visible liquid using a P10 pipette tip
▢ Air-dry the beads pellet for 2min (until the beads pellet is dry)
👉 Do not over-dry the beads pellet to not reduce DNA recovery. The beads must still dark brown and glossy looking ; the beads must not turn lighter brown and start to crack
▢ Remove the 0.2ml tube from the magnetic stand
▢ Add 22µl of Elution Buffer (white) to the beads pellet
▢ Mix well by pipetting up and down 10 times (until homogenization)
▢ If necessary, quickly spin the sample to collect the liquid from the sides of the 0.2ml tube
▢ Incubate 2min at room temperature
▢ Place the 0.2ml tube against an appropriate magnetic stand for 3min (until the solution is clear) to separate the beads from the supernatant
▢ Transfer 20µl of the clear supernatant containing the indexed DNA library to a new 0.2ml PCR tube
📝 Make sure not to disturb the beads pellet
🔴 Safe stopping point : Sample can be stored overnight at -20°C
4.3. QC Libraries (Figure 1)
4.3.1. Quantification is performed on a Qubit 3.0 fluorometer with DS DNA Broad Range Quantitation Assay kit according manufacturer’s recommendations (Agilent, ThermoFisher Scientific)
Average concentration per library should be approximately 30-50ng/µl
4.3.2. Validation is performed on a 2100 Bioanalyzer with DNA 7500 kit according manufacturer’s recommendations (Agilent Technologies) (Figure 2)
📝 Average size distribution should be approximately 370-420bp2
📝 132 adaptor nucleotides shift between insert and library size
Twist Bioscience Targeted Methylation Sequencing Protocol (DOC-001222 REV 4.02)
👉 All reagents thaw on ice unless otherwise specified, then pulse-vortex 2sec to mix and pulse-spin
5. Pooling of 8 samples (Figure 1)
🔖 Total DNA amount per pool depends on the number of libraries in the pool
🔖 Total DNA amount per pool of 8 samples should be 1500ng
🔖 The amount of each library per pool will be the same
▢ Transfer the calculated volumes from each indexed library in a PCR 0.2ml tube appropriate for the hybridization reaction later
▢ Add the following reagents to the pool of 8 indexed libraries :
▢ Twist Bioscience Custom Methylation Panel (0,01fmol/probe 2) 4µl
▢ Universal Blocker 8µl
▢ Blocker Solution (human Cot-1) 5µl
▢ Methylation Enhancer 2 custom 2µl
▢ Mix by flicking the 0.2ml tube
▢ Perform a quick spin
👉 Ensure there are minimal bubbles present
▢ Dry the pre-hybridization solution using a vacuum concentrator into 0.2ml appropriate support at low heat (30°C)
🔴 Safe stopping point : Dry pre-hybridization solution can be stored overnight at -20°C
6. Hybridization (Figure 1)
▢ Pre-heat a Thermal Cycler at 95°C
▢ Pre-heat a Thermal Cycler at 65°C
▢ Thaw Fast Hybridization Mix at room temperature
▢ Thaw Hybridization Enhancer at room temperature
▢ Heat 22µl of Fast Hybridization Mix at 65°C for 10min (for each pool)
📝 Do not allow the Fast Hybridization Mix to cool to room temperature
▢Resuspend the dried pre-hybridization material with 20µl pre-heated Fast Hybridization Mix
👉 Fast Hybridization Mix is viscous, pipette slowly to ensure accuracy
📝 The presence of small particles in the custom methylation panel will not interfere with performance
▢ Perform a quick spin
👉 Ensure there are no bubbles present
▢ Place the 0.2ml tube of pre-hybridization solution in the pre-heated 65°C Thermal Cycler
▢ Add 30µl of Hybridization Enhancer to the top of the pre-hybridization solution
📝 Hybridization enhancer is mineral oil to prevent evaporation
▢ Perform a quick spin
📝 Hybridization enhancer settles on top of the hybridization reaction does not affect the final captured product
▢ Place the 0.2ml tube of hybridization solution in the preheated 95°C Thermal Cycler and run the following program :
Temperature Time
95°C 5min
60°C2 custom 16hr2 custom
👉 Make sure the 0.2ml tube is sealed tightly to prevent evaporation during the incubation
7. Capture (Figure 1)
7.1. Streptavidin beads preparation (Figure 1)
▢ Equilibrate Streptavidin Binding beads to room temperature (at least 30min before use)
▢ Equilibrate Fast Binding Buffer to room temperature
▢ Vortex Streptavidin Binding Beads until mixed well
▢ For each capture reaction, add 100µl Streptavidin Binding Beads to a clean 1.5ml tube (1 tube per hybridization)
▢ Add 200µl Fast Binding Buffer
▢ Mix by pipetting until mixed well
▢ Place on a compatible magnetic stand
▢ Incube 1min at room temperature
▢ Carefully remove and discard supernatant
📝 Do not disturb the bead pellet
▢ Remove from the magnetic stand
▢ Add 200µl Fast Binding Buffer
▢ Mix by pipetting until mixed well
▢ Place on a compatible magnetic stand
▢ Incube 1min at room temperature
▢ Carefully remove and discard supernatant
📝 Do not disturb the bead pellet
▢ Remove from the magnetic stand
▢ Add 200µl Fast Binding Buffer
▢ Mix by pipetting until mixed well
▢ Place on a compatible magnetic stand
▢ Incube 1min at room temperature
▢ Carefully remove and discard supernatant
📝 Do not disturb the bead pellet
▢ Remove from the magnetic stand
▢ Add 200µl Fast Binding Buffer
▢ Resuspend washed beads by vortexing until homogenized
7.2. Streptavidin binding beads (Figure 1)
▢ After hybridization is complete, quickly and directly transfer the full volume (including hybridization enhancer) of an hybridization into a 1.5ml tube of washed streptavidin beads
⚠ Rapid transfer directly from the Thermal Cycler is a critical step for minimizing off-target binding
▢ Mix by pipetting and flicking
▢ Mix on a rotator mixer for 30min at room temperature at a sufficient speed to keep the solution mixed (22 rpm)
7.3. Wash non specific captured targets (Figure 1)
▢ If a precipitate is observed, heat at 48°C Fast Binding Buffer, Fast Wash Buffer 1 and Wash Buffer 2 until all precipitate is dissolved
▢ Pre-heat 500µl Fast Wash Buffer 1 to 63°C2 custom in a thermomixer (for each pool, 1 pool per 1.5ml tube)
▢ Pre-heat 700µl Wash Buffer 2 to 48°C in a thermomixer (for each pool, 1 pool per 1.5ml tube)
▢ Remove the 1.5ml tube containing the hybridization reaction with streptavidin binding beads from the rotator mixer
▢ Perform a quick spin to ensure that the whole solution is at the bottom of the 1.5ml tube
▢ Place on a compatible magnetic stand
▢ Incubate 1min at room temperature
▢ Carefully remove and discard supernatant including hybridization enhancer
📝 Do not discard beads pellet that contain DNA targets
📝 Trace amount of hybridization enhancer may be visible after supernatant removal, but it will not affect the final capture product
▢ Remove the 1.5ml tube from the magnetic stand
▢ Add 200µl preheated 63°C 2 custom Fast Wash Buffer 1
▢ Mix by pipetting
▢ Perform a quick spin
▢ Incubate 5min at 63°C in the pre-heated thermomixer
▢ Place the 1.5ml tube on a compatible magnetic stand
▢ Incube 1min at room temperature
▢ Carefully remove and discard clear supernatant
📝 Do not discard beads pellet that contain DNA targets
▢ Remove the 1.5ml tube from the magnetic stand
▢ Add 200µl preheated 63°C 2 custom Fast Wash Buffer 1
▢ Mix by pipetting
▢ Perform a quick spin
▢ Incubate 5min at 63°C in the pre-heated thermomixer
▢ Perform a quick spin to ensure all solution is at the bottom of the 1.5ml tube
▢ Transfer the entire volume to a new 1.5ml tube
👉 This step reduces background resulting from non-specific binding to the surface of the 1.5ml tube
▢ Place the 1.5ml tube on a compatible magnetic stand
▢ Incubate 1min at room temperature
▢ Carefully remove and discard clear supernatant
📝 Do not discard beads pellet that contain DNA targets
▢ Remove the 1.5ml tube from the magnetic stand
▢ Add 200µl preheated 48°C Wash Buffer 2
▢ Mix by pipetting
▢ Perform a quick spin
▢ Incubate 5min at 48°C in the pre-thermomixer
▢ Place the 1.5ml tube on a compatible magnetic stand
▢ Incubate 1min at room temperature
▢ Carefully remove and discard clear supernatant
📝 Do not discard beads pellet that contain DNA targets
▢ Add 200µl preheated 48°C Wash Buffer 2
▢ Mix by pipetting
▢ Perform a quick spin
▢ Incubate 5min at 48°C in the pre-heated thermomixer
▢ Place the 1.5ml tube on a compatible magnetic stand
▢ Incubate 1min at room temperature
▢ Carefully remove and discard clear supernatant
📝 Do not discard beads pellet that contain DNA targets
Add 200µl preheated 48°C Wash Buffer 2
▢ Mix by pipetting
▢ Perform a quick spin
▢ Incubate 5min at 48°C in pre-heated thermomixer
▢ Place the 1.5ml tube on a compatible magnetic stand
▢ Incubate 1min at room temperature
▢ Carefully remove and discard clear supernatant
📝 Do not discard beads pellet that contain DNA targets
▢ Remove all traces of supernatant using a 10µl pipette tip
📝 before pipetting, the beads pellet may be briefly spun to collect supernatant at the bottom of the 1.5ml tube and returned to the magnetic stand
👉 Proceed immediately to the next step, do not allow the beads to dry
▢ Remove the 1.5ml tube from the magnetic stand
▢ Add 45µl molecular biology grade water
▢ Mix by pipetting until homogenization
▢ Place this solution of streptavidin binding beads slurry on ice
🔴 Safe stopping point : slurry can be stored overnight at -20°C, possible stop point
8. Amplification (Figure 1)
8.1. PCR amplification (Figure 1)
▢ Mix by pipetting streptavidin binding beads slurry
▢ Transfer 22.5µl streptavidin binding beads slurry to a new 0.2ml tube appropriate to Thermal Cycler
👉 Store the remaining 22.5µl streptavidin binding beads slurry at -20°C for future use
▢ On ice, add the following reagents to the 0.2ml tube containing streptavidin binding beads slurry :
▢ P5P7 Primers Mix (10µM) : 2.5µl
▢ Equinox Library Amp Mix2 (2X) : 25µl
Total volume : 50µl
▢ Mix gently by pipetting
▢ Perform a quick spin to collect all liquid from the sides of the 0.2ml tube
▢ Place the 0.2ml tube in a thermocycler and run the following program :
Cycles step Temperature Time Cycles 2 custom
Initial denaturation 98°C 45sec 1
Denaturation 98°C 15sec
Annealing 60°C 30sec 10
Extension 72°C 30sec
Final extension 72°C 1min 1
Hold 4°C
👉 Proceed immediately to the next step
8.2. Clean-up of amplified library2 (Figure 1)
▢ Equilibrate purification beads to room temperature (30min)
▢ Vortex purification beads until mixed well
▢ Add 90µl (1.8X) of homogenized beads to each library
▢ Mill well by vortexing (until homogenization)
📝 It’s not necessary to recover supernatant or remove the streptavidin binding beads from the amplified product
▢ Incubate 5min at room temperature
▢ Place 0.2ml tube against an appropriate magnetic stand for 5min (or when the solution is clear) to separate the beads from the supernatant
▢ Carefully remove and discard supernatant
📝 Do not discard beads pellet that contain DNA targets
▢ Add 200µl of 80% freshly prepared 80% ethanol to gently wash the beads pellet
📝 Do not disturb the bead pellet
▢ Incubate 1min
▢ Carefully remove and discard ethanol
📝 Do not discard beads that contain DNA targets
▢ Add 200µl of 80% freshly prepared 80% ethanol to gently wash the beads pellet
📝 Do not disturb the beads pellet
▢ Incubate 1min
▢ Carefully remove and discard ethanol
📝 Do not discard beads that contain DNA targets
▢ Remove all traces of ethanol using a 10µl pipette tip
▢ Before pipetting, the beads pellet may be briefly spun to collect ethanol at the bottom of the 0.2ml tube and returned to the magnetic stand
▢ Air-dry the beads pellet for 2min (or until the beads pellet is dry)
📝 Do not over-dry the beads pellet
▢ Remove the 0.2ml tube from the magnetic stand
▢ Add 32µl of water to the beads pellet
▢ Mix well by pipetting up and down 10 times (until homogenization)
▢ If necessary, quickly spin the sample to collect the liquid from the sides of the 0.2ml tube
▢ Incubate 2min at room temperature
▢ Place the 0.2ml tube against an appropriate magnetic stand for 3min (until the solution is clear) to separate the beads from the supernatant
▢ Transfer 30µl of the clear supernatant containing the targets DNA to a new PCR 0.2ml tube
📝 Make sure not to disturb the beads pellet
🔴 Safe stopping point : Sample can be stored overnight at -20°C
8.3. QC Library (Figure 1)
8.3.1. Quantification is performed on a Qubit 3.0 fluorometer with DS DNA High Sensitivity Quantitation Assay kit according manufacturer’s recommendations (Agilent, ThermoFisher Scientific)
Average concentration of libraries’s pool should be approximately 5-30ng/µl
8.3.2. Validation is performed on a 2100 Bioanalyzer with DNA 7500 kit according manufacturer’s recommendations (Agilent Technologies) (Figure 3)
📝 Average size distribution should be approximately 370-420bp 2
Protocol2 is the functional protocol, after adapted of protocol1. Differences between protocol1 and protocol2 are listed below :
· Steps 1 to 4 title : Size library protocol : 470-520bp1 => 370-420bp2
· Step 1.1. : Input DNA : 200ng1 => 50ng or 100ng2
· Step 1.1. : Covaris ultrasonication parameters 1 with M220 Covaris :
Peak Incident Power (W) = 75
Duty Factor (%) = 10
Cycles per Burst (cpb) = 200
Time (sec) = 75sec
· Step 1.1. : Size fragmentation : 350-400bp1=> 240-290bp2
· Step 4.1. : Number of indexing cycles : 6 cycles 1 => 11 cycles or 9 cycles 2
· Step 4.2. : Purification beads for clean up indexed library : 0.6X 1 => 0.9X 2
· Step 4.3.2. : Size converted library : 470-520bp1 => 370-420bp2
· Title steps 5 to 8 : Protocol version : DOC-001066 REV. 1.01 =>DOC-001222 REV 4.02
· Step 5. : 0,22fmol/probe/4µl 1 => 0,01fmol/probe/4µl 2
· Step 5. : Hybridization without Methylation Enhancer 1 => Methylation Enhancer 2
· Step 6. : Hybridization temperature : 65°C1 => 60°C2
· Step 6. : Hybridization time : 4hrs1 => 16hrs2
· Step 3. : Wash Buffer 1 Temperature : 70°C 1 => 63°C 2
· Step 8.1. : Enzyme of amplification : KAPA HiFi HotStart ReadyMix 1 (Roche Diagnostic) => Equinox Library Amp Mix2 (Twist Bioscience)
· Step 8.1. : Number of amplification cycles : 9 cycles 1 => 10 cycles 2
· Step 8.2. : Two Clean-up amplified library1 (1.8X et 0.8X) => One Clean-up amplified library2 (1.8X)
· Step 8.3.2. : Size captured library : 470-520bp 1 => 370-420bp 2