NEXTflex™ mtDNA-Seq for cell samples
Briefly spin down each component to ensure material has not lodged in the cap or side of tube.
Keep on ice and vortex each tube prior to use.
Allow Agencourt AMPure XP Beads to come to room temperature and vortex the beads until liquid appears homogenous before every use.
For each sample, combine the following reagents on ice in nuclease-free microcentrifuge tubes: 
μLNuclease-free Water_ μLgDNA (4 – 8 μg)5 μLNEXTflex™ mtDNA Buffer Mix 15 μLNEXTflex™ mtDNA Buffer Mix 25 μLNEXTflex™ Nuclear DNA Digest Mix50 μLTOTAL. 
Mix well by pipetting.
Incubate in a heat block for 48 hours at 37°C.
Incubate the sample at 70°C for 30 minutes.
Spin the tube for 10 seconds to collect contents of the tube.
Add 50 μL of AMPure XP Beads to each sample and mix well by pipetting.
Incubate at room temperature for 5 minutes.
Place the tube on the magnetic rack at room temperature for 5 minutes or until thesupernatant appears clear.
Remove and discard clear supernatant taking care not to disturb beads.
Some liquidmay remain in the tube.
Wash #1: With tube on rack, gently add 200 μL of freshly prepared 80% ethanol to each magnetic bead pellet and incubate plate at room temperature for 30 seconds.
Carefully, remove ethanol by pipette.
Wash #2: With tube on rack, gently add 200 μL of freshly prepared 80% ethanol to each magnetic bead pellet and incubate plate at room temperature for 30 seconds.
Carefully, remove ethanol by pipette.
Ensure all ethanol has been removed.
Remove the tube from the magnetic rack and let dry at room temperature for 3 minutes.
Do not overdry the beads.
Resuspend dried beads with 36 μL Nuclease-free Water. 
Mix well by pipetting. 
Ensure beads are no longer attached to the side of the well. 
Incubate resuspended beads at room temperature for 2 minutes.
Place tube on magnetic rack for 5 minutes or until the sample appears clear.
Gently transfer 35 μL of clear sample to a fresh microcentrifuge tube.
To ensure complete removal of nuclear DNA contamination, repeat the digestion with 35 μL of eluted material from Step 18.
Set up the reaction as follows:
35 μLEluted DNA (Step 18)5 μLNEXTflex™ mtDNA Buffer Mix 15 μLNEXTflex™ mtDNA Buffer Mix 25 μLNEXTflex™ nuclear DNA Digest Mix50 μLTOTAL. 
Incubate for 2 hours at 37°C.
Repeat steps 6-18.
For each sample prepare two separate reactions in adjacent wells of a 96-well PCR Plate on ice as described below: 
Mix well by pipetting.
Apply adhesive PCR plate seal and place in thermocycler for the following PCR cycles: 
Prepare pre-stained SYBR Gold 2% or Ethidium Bromide TAE agarose gel.
Mix and pour into gel tray.
Load 3 μL of the PCR product mixed with 2 μL of 6X Loading Dye and 7 μL of Nuclease-free Water.
Load the samples on the gel along with 6 μL of MW 100 bp Ready-to-Load Ladder.
Run the gel with 1X TAE buffer at 100-120V for 60-120 minutes, or until bands have adequately resolved.
Visualize the gel on UV transilluminator or gel documentation instrument.
Check for the presence of a mtDNA band and absence/significant reduction (compared to the control) of nuclear DNA bands to proceed with the subsequent steps (Fig. 2 in Guidelines).
Adjust mtDNA sample volume to 130 μL with Nuclease-free Water.
Set pipette to 130 μL and mix well by pipetting.
Transfer the sample to a Covaris tube.
Follow manufacturer's instructions.
For example, using the Covaris S2 system, the following parameters will produce fragments of 150-200 bp Peak Intensity – 5 Duty cycle – 10% Cycles per burst – 200 Time – 180 s. 
Transfer your sample from a Covaris tube to a 15 mL microcentrifuge tube.
Option 1: SpeedVac - following sonication, spin down sample in a SpeedVac for 2 hoursat 45°C to reduce the volume of your sample to ≤ 40 μL.
Do not let the sample drydown completely.
Option 2: Bead Cleanup – Alternatively a 2X AMPure XP bead clean up can be done as follows: 
After concentrating the sample, Qubit® dsDNA reagents can be used to quantify the DNA concentration.
For each sample, combine the following reagents on ice in a nuclease-free 96 well PCR Plate: 
40 μLmtDNA (from section "Fragmentation of mtDNA")7 μLNEXTflex™ mtDNA End Repair Buffer Mix3 μLNEXTflex™ mtDNA End Repair Enzyme Mix50 μLTOTAL. 
Mix well by pipetting.
Apply adhesive PCR plate seal and incubate on a thermocycler for 30 minutes at 22°C.
Add 80 μL of AMPure XP Beads to each sample and mix well by pipetting.
Incubate sample at room temperature for 5 minutes.
Place the 96 well PCR Plate on the magnetic stand at room temperature for 5 minutes or until the supernatant appears clear.
Remove and discard clear supernatant taking care not to disturb beads.
Some liquid may remain in wells.
Wash #1: With plate on stand, gently add 200 μL of freshly prepared 80% ethanol to each magnetic bead pellet and incubate plate at room temperature for 30 seconds.
Carefully, remove ethanol by pipette.
Wash #2: With plate on stand, gently add 200 μL of freshly prepared 80% ethanol to each magnetic bead pellet and incubate plate at room temperature for 30 seconds.
Carefully, remove ethanol by pipette.
Ensure all ethanol has been removed.
Remove the plate from the magnetic stand and let dry at room temperature for 3 minutes.
Note: Do not overdry the beads.
Resuspend dried beads with 17 μL Resuspension Buffer.
Mix well by pipetting.
Ensure beads are no longer attached to the side of the well.
Incubate resuspended beads at room temperature for 2 minutes.
Place plate on magnetic stand for 5 minutes or until the sample appears clear.
Gently transfer 16 μL of clear sample to a new well.
Combine the following in the 96 well PCR Plate:
16 μLEnd-Repaired DNA (from section "Clean-Up")4.5 μLNEXTflex™ mtDNA Adenylation Mix20.5 μLTOTAL. 
Mix well by pipetting.
Apply adhesive PCR plate seal and incubate on a thermocycler for 30 minutes at 37°C.
For each sample, combine the following reagents (in this order) in the 96-well PCR Plate:
20.5 μL3’ Adenylated DNA (from the above section)27.5 μLNEXTflex™ mtDNA Ligation Mix2.0 μLNEXTflex™ mtDNA Adapter or NEXTflex™ ChIP Barcode50 μLTOTAL. 
Mix well by pipetting.
Apply adhesive PCR plate seal and incubate on a thermocycler for 15 minutes at 22°C.
Add 40 μL of AMPure XP Beads to each sample and mix well by pipetting.
Incubate at room temperature for 5 minutes.
Place the 96 well PCR Plate on the magnetic stand at room temperature for 5 minutes or until the supernatant appears clear.
Set pipette to 88 μL and gently remove clear supernatant taking care not to disturbbeads.
Some liquid may remain in wells.
Wash #1: With plate on stand, gently add 200 μL of freshly prepared 80% ethanol to each sample and incubate plate at room temperature for 30 seconds.
Carefully, remove ethanol by pipette.
Wash #2: With plate on stand, gently add 200 μL of freshly prepared 80% ethanol to each sample and incubate plate at room temperature for 30 seconds.
Carefully, remove ethanol by pipette.
Ensure all ethanol has been removed.
Remove the plate from the magnetic stand and let dry at room temperature for 3 minutes.
Resuspend dried beads with 52 μL Resuspension Buffer.
Mix well by pipetting and ensuring beads are no longer attached to the side of the well.
Incubate resuspended beads at room temperature for 2 minutes.
Place plate on magnetic stand for 5 minutes or until the sample appears clear.
Gently transfer 50 μL of clear sample to new well.
Add 40 μL of AMPure XP Beads to each sample and mix well by pipetting.
Incubate at room temperature for 5 minutes.
Place the 96 well PCR Plate on the magnetic stand at room temperature for 5 minutes or until the supernatant appears clear.
Set pipette to 88 μL and gently remove clear supernatant taking care not to disturb beads.
Some liquid may remain in wells.
Wash #1: With plate on stand, gently add 200 μL of freshly prepared 80% ethanol to each sample and incubate plate at room temperature for 30 seconds.
Carefully, remove ethanol by pipette.
Wash #2: With plate on stand, gently add 200 μL of freshly prepared 80% ethanol to each sample and incubate plate at room temperature for 30 seconds.
Carefully, remove ethanol by pipette.
Ensure all ethanol has been removed.
Remove the plate from the magnetic stand and let dry at room temperature for 3 minutes.
Resuspend dried beads with 38 μL Resuspension Buffer. 
Mix well by pipetting Ensure beads are no longer attached to the side of the well.
Incubate resuspended beads at room temperature for 2 minutes.
Place plate on magnetic stand for 5 minutes or until the sample appears clear.
Gently transfer 36 μL of clear sample to new well.
For each sample, combine the following reagents on ice in the 96 well PCR plate:
36 μLPurified Ligation Product (from section "Clean-Up 2")12 μLNEXTflex™ PCR Master Mix2 μLNEXTflex™ Primer Mix50 μLTOTAL. 
Mix well by pipetting.
PCR Cycles: Add 40 μL of AMPure XP Beads to each sample and mix well by pipetting.
Incubate at room temperature for 5 minutes.
Place the 96 well PCR Plate on the magnetic stand at room temperature for 5 minutesor until the supernatant appears clear.
Set pipette to 88 μL and gently remove clear supernatant taking care not to disturbbeads.
Some liquid may remain in wells.
Wash #1: With plate on stand, gently add 200 μL of freshly prepared 80% ethanol to each sample and incubate plate at room temperature for 30 seconds.
Carefully, remove ethanol by pipette.
Wash #2: With plate on stand, gently add 200 μL of freshly prepared 80% ethanol to each sample and incubate plate at room temperature for 30 seconds.
Carefully, remove ethanol by pipette.
Ensure all ethanol has been removed.
Remove the plate from the magnetic stand and let dry at room temperature for 3 minutes.
Resuspend dried beads with 52 μL Resuspension Buffer.
Mix well by pipetting and ensuring beads are no longer attached to the side of the well.
Incubate resuspended beads at room temperature for 2 minutes.
Place plate on magnetic stand for 5 minutes or until the sample appears clear.
Gently transfer 50 μL of clear sample to new well.
Add 40 μL of AMPure XP Beads to each sample and mix well by pipetting.
Incubate at room temperature for 5 minutes.
Place the 96 well PCR Plate on the magnetic stand at room temperature for 5 minutesor until the supernatant appears clear.
Set pipette to 90 μL and gently remove clear supernatant taking care not to disturbbeads.
Some liquid may remain in wells.
Wash #1: With plate on stand, gently add 200 μL of freshly prepared 80% ethanol to each sample and incubate plate at room temperature for 30 seconds.
Carefully, remove ethanol by pipette.
Wash #2: With plate on stand, gently add 200 μL of freshly prepared 80% ethanol to each sample and incubate plate at room temperature for 30 seconds.
Carefully, remove ethanol by pipette.
Ensure all ethanol has been removed.
Remove the plate from the magnetic stand and let dry at room temperature for 3 minutes.
Resuspend dried beads with 16 μL Resuspension Buffer.
Mix well by pipetting.
Ensurebeads are no longer attached to the side of the well.
Incubate resuspended beads at room temperature for 2 minutes.
Place plate on magnetic stand for 5 minutes or until the sample appears clear.
Gently transfer 15 μL of clear sample to a well of a new 96 well PCR Plate.
qPCR is recommended to quantitate DNA library templates for optimal cluster density.
