Adenylate 3ʹ Ends

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This step adds an adenine (A) nucleotide to the 3ʹ ends of the blunt fragments to prevent them from ligating to each other. A corresponding thymine (T) nucleotide on the 3ʹ end of the adapter provides a complementary overhang for ligating the adapter to the fragment.

• ATL4 (A-Tailing Mix)
• Microseal 'B' adhesive film
• Prepare for a later procedure:
– Anchor plate (RNA Index Anchors)
– STL (Stop Ligation Buffer)
1. Prepare the following consumables:

Reagent

Storage

Instructions

Anchor plate

-25°C to -15°C

Thaw at room temperature. Vortex to mix, and then centrifuge briefly.

ATL4

-25°C to -15°C

Thaw at room temperature. Flick to mix, and then centrifuge briefly.

STL

-25°C to -15°C

Thaw at room temperature. Vortex to mix, and then centrifuge briefly.
2. Save the following ATAIL program on the thermal cycler:
• Choose the preheat lid option and set to 100°C
• Reaction volume is 30 µl
• 37°C for 30 minutes
• 70°C for 5 minutes
• Hold at 4°C
1. If you are resuming the protocol after a safe stopping point, centrifuge the sealed PCR plate at 280 × g for 10 seconds.
2. Add 12.5 µl ATL4 to each well.
3. Using a 200 µl pipette, pipette 10 times to mix, and then seal.
4. Place on the preprogrammed thermal cycler and run the ATAIL program.

Total program time is ~38 minutes and each well contains a volume of 30 µl.