TaME-seq2

Prepare and normalize DNA/cDNA samples by measuring sample concentration and diluting in nuclease-free water if necessary.

Sample volume should be 15µL

50ng input is recommended, but the protocol works with less and performance is more dependent on viral load.

Prepare a master mix for the tagmentation reaction and add sample DNA and master mix to individual wells.

A	B	C
Reagent	1x	10x
Bead linked transposoms (BLT)	5 μL	50 μL
Tagmentation buffer 1 (TB1)	5 μL	50 μL
Sample DNA/cDNA	15 μL
Total	25 μL	10x10 μL

Tagmentation reagent mix table

Incubate the samples as follows:

0h 15m 0s at 55°C

Hold at 4°C

Add 5µL of Tagmentation stop buffer (TSB) to each sample.

Incubate sample for 0h 15m 0s at 37°C

Wash the samples

1. Place tubes on the magnetic rack for 0h 3m 0s (or until solution is clear)
2. Discard supernatant
3. Remove tube from magnetic rack and add 50µL Tagment wash buffer (TWB) and mix to resuspend beads
4. Place tubes on magnetic rack for 0h 3m 0s and remove supernatant
5. Repeat step 3 and 4 for a total of 2 washes
6. Add 50µL TWB to samples and mix
7. Close cap on tubes and place on magnet. Allow to incubate for at least 0h 3m 0s and continue the protocol. The samples will be used later in the PCR
   Note

   The samples are submerged in TWB to stop the beads from overdrying while working on the next steps. The samples can also be eluted in 14µL nuclease-free water and stored in the freezer, if so do not add H2O/elution buffer to the PCR master mix in the next step.

1. Make the PCR master mix | A | B | C | | --- | --- | --- | | Reagent | 1x | 10x | | 2x PCR master mix | 12,5 μl | 125 μl | | Q-solution x5 | 2,5 μl | 25 μl | | Primer pool (15 μM) | 1 μl | | | i5/i7 indexes (10 μM) | 2 μl | | | Nuclease-free water | 7 μl | 70 μl | | Total | 25 μl | 22 μL x 10 μL + 3 μL primers per sample |

2. Remove supernatant from samples prepared in step 6.7 and remove from magnetic rack

3. Add 30µL of PCR MM to the samples, mix well and resuspend the beads (the beads can be difficult to resuspend, but they don't need to be completely respuspended)

4. Pipette 15µL out of the sample to a new well containing the nuclease-free water, primer pool and i5/i7 indexes, for seperate F and R reactions

5. Run the touchdown PCR reaction using the following program on the thermal cycler:

      **Step**                      **Temperature**                          **Time**              **Cycles** 
   

Initial denaturation 95°C 0h 5m 0s 1

Touchdown PCR:

Denaturation 95°C 0h 0m 30s 10

Annealing 68°C-58°C 0h 1m 30s 10 (decrease 1°C per cycle)

Extension 72°C 0h 0m 30s 10

PCR:

Denaturation 95°C 0h 0m 30s 26

Annealing 58°C 0h 1m 30s 26

Extension 72°C 0h 0m 30s 26

Final extension 68°C 0h 10m 0s 26

Hold 4°C

1. After the PCR reaction is finished place the plate on the magnet for 0h 5m 0s

2. Transfer 20µL of the supernatant to a fresh well (transfer less than the total volume to compensate for differences in volume during pipetting).

3. Pool 10µL of each sample in an eppendorf tubes.

4. Vortex and invert purification beads (PB) to fully resuspend.

5. Prepare at master mix of diluted PB (Note: The volumes used in the master mix is for each sample that has been pooled).

A	B
Reagent	Volume per reaction
PB	10 µL
Nuclease-free water	8,85 µL

6. Vortex and mix the diluted master mix and add 18.75µL (per reaction) master mix to the pool and mix well

7. Seal the tubes and incubate at room temperature for 0h 5m 0s

8. Place on magnet for 0h 5m 0s 5 min or until clear

9. Add 3.7µL (per reaction) of PB to a new eppendorf tube

10. Transfer 27µL (per reaction) of the supernatant to the eppendorf tube containing PB and mix well

11. Seal the tube and incubate at room temperature for 0h 5m 0s

12. Place the tube on a magnet for 0h 5m 0s or until clear

13. Remove and discard the supernatant without disrupting the beads

14. With the tube on the magnet, add fresh 80% ethanol to cover the beads without mixing and incubate 0h 1m 0s

15. Remove the ethanol

16. Repeat steps 14 and 15 for a total of 2 washes

17. Remove any excess liquid from the tube

18. Air-dry on the magnet for 0h 5m 0s or until dry. The bead should not dry so long that it cracks while ethanol residues should have evaporated.

19. Remove the tube from the magnet and add 205µL (more or less depending on desired reaction volume to elute in) resuspension buffer/water-free nuclease to the beads and mix

20. Incubate at room temperature for 0h 5m 0s

21. Place tube on magnet for 0h 2m 0s or until clear

22. Transfer 200µL of the supernatant into a fresh tube

23. Clean up two more times (start from step 12) using 0,65x ratio Ampure beads, elute in 42µL and transfer 40µL to a new tube before assessing size distribution again.

Assess the quality of the pools using a Bioanalyzer or an equivalent instrument to see the fragment size distribution.

If there is an excess of small fragments in the library, clean up one or more times (start from step 11) using 0,65x ratio Ampure beads, elute in 42µL and transfer 40µL to a new tube before assessing size distribution again.