HTTM : Illumina libraries

Transfer2.5µL of DNA from the DNA extraction plate to a new PCR plate.

Prepare a fragmentation master mix with :

A	B
NEB Ultra II FS buffer	77 µl
NEB Ultra II FS enzyme	22 µl
Molecular grade water	11 µl

Add 1µL of the fragmentation master mix to each well.

Incubate in a thermocycler with the following protocol :

• 0h 15m 0sat 37°C
• 0h 30m 0sat 65°C

Add 1µLof 4µM Nextera (NxT) adaptors to each well.

Prepare a ligation master mix with :

A	B
NEB Ultra II ligation master mix	377.4 µl
NEB Ultra II ligation enhancer	12.1 µl

Add 3.5µLof ligation master mix to each well.

Incubate in a thermocycler with the following protocol :

• 0h 30m 0sat 20°C
• 0h 10m 0sat 65°C

Prepare a PCR master mix with :

A	B
NxT_A primer 20 µM	880 µl
Nxt_B primer 20 µM	880 µl
Molecular grade water	8360 µl
PCR Supermix 2X	11000 µl

Add 192µLof PCR master mix to each well.

Split the PCR reaction into 4 different plates (50µl per plate).

Incubate each plate in a thermocycler with the following cycles :

• 0h 0m 30sat 98°C
• 0h 0m 15sat 98°C
• 0h 0m 30sat 72°C
• Repeat from step 2 for 20~25 cycles*
• 0h 2m 0s 72°C

Pool the 4 PCR replicates together in a PCR plate.

Transfer2µLof DNA from the pool plate to a new PCR plate.

Add 2µLof each barcoding primer to the DNA :

• Nxt_i5_barcoding

• Nxt_i7_barcoding

Prepare a PCR master mix with :

A	B
Molecular grade water	2090 µl
PCR supermix 2X	2750 µl

Add 44µL of the PCR master mix to each well of the plate.

Incubate in a thermocycler using the following protocol :

• 0h 0m 30s at 98°C
• 0h 0m 15s at 98°C
• 0h 1m 0s at 72°C (no anneal step)
• Repeat from step 2 for 5 cycles
• 0h 2m 0s at 72°C

Pool together 2µL of each sample.

Purify with Ampure XP SPRI beads using a 0.8 ratio. Resuspend with50µL of molecular grade water.

Proceed with QC and sequencing.