Ligate Anchors

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This step ligates pre-index anchors to the ends of the double-stranded cDNA fragments to prepare them for dual indexing. A subsequent PCR amplification step adds the index adapter sequences.

• LIGX (Ligation Mix)
• STL (Stop Ligation Buffer)
• Anchor plate (RNA Index Anchors)
• RSB (Resuspension Buffer)
• Microseal 'B' adhesive film
• Prepare for a later procedure:
– Agencourt AMPure XP
• Although stored at -25°C to -15°C, LIGX remains liquid and does not require thawing.
• Each well of the anchor plate is single-use and contains RNA Index Anchors. Anchor plate wells contain the same content and can be used in any order.
• The anchor plate is unique from the index plate, which is used in a later step. Check the plate labels to make sure you are using the correct plate.
1. Prepare the following consumables:

Reagent

Storage

Instructions

AMPure XP

2°C to 8°C

Let stand for 30 minutes to bring to room temperature. Vortex and invert to mix.

LIGX

-25°C to -15°C

Store until needed. Flick to mix, and then centrifuge briefly.

RSB

-25°C to -15°C

Thaw at room temperature. Vortex and invert to mix.
2. Save the following LIG program on the thermal cycler:
• Choose the preheated lid option and set to 100°C
• Reaction volume is 38 µl
• 30°C for 10 minutes
• Hold at 4°C

Add Anchors

1. Centrifuge the sealed PCR plate at 280 × g for 10 seconds.
2. Add the following volumes to each well in the order listed. Transfer RNA Index Anchors from the anchor plate to the PCR plate.

Order of Addition

Reagent

Volume for Sample Input ≤ 100 ng (µl)

Volume for Sample Input > 100 ng (µl)

1

RSB

2.5

0

2

RNA Index Anchors

2.5

5

3

LIGX

2.5

2.5
3. Using a 200 µl pipette, pipette 10 times to mix, and then seal.
4. Place on the preprogrammed thermal cycler and run the LIG program.

Total program time is ~13 minutes and each well contains a volume of 38 µl.

Stop Ligation

1. Centrifuge the sealed PCR plate at 280 × g for 10 seconds.
2. Add 5 µl STL to each well.
3. Pipette 15 times to mix.

Each well contains a volume of 43 µl.