Amplify Library

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Choose and record a unique index mix for each sample to ensure that no sample index combinations overlap in a multiplexed sequencing run.

Align the orientation of the hole on the side of the strip containing the indexes with the index map in the box lid and in the following diagram:

Pierce the foil with a 20 µl pipette before aspirating. Check orientation of index tube strip to ensure that the correct index combination is added and recorded for sequencing. There are eight index mixes provided with this kit to allow for unique dual indexing of eight samples.

To pool more than eight libraries together in a sequencing run, use the Illumina Single Cell Unique Dual Indexes plate. For more information on pooling, refer to Index Adapters Pooling Guide. For more information on recommended indexes, refer to Illumina Adapter Sequences.

Preparation

Prepare the following consumables:

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PMM4—Pipette 10 times, and then centrifuge briefly.

Save the following PCR program on the thermal cycler:

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Set the lid temperature to 105°C

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98°C for 45 seconds

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X cycles of:

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98°C for 15 seconds

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67°C for 30 seconds

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69°C for 45 seconds

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72°C for 1 minute

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Hold at 4°C

Adjust the program to optimize for different input quality and concentration.

Sample Input	Number of PCR Cycles (X)
High RNA sample types (eg, cell lines)	10
Low RNA sample types (eg, primary cells and nuclei)	13

Decrease by 1–2 cycles for cDNA QC concentrations > 200 ng/µl.

Procedure

Add the following volumes to each reaction tube, in the order listed:

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Unique Library Index Mix (5 µl)

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PMM4 (12.5 µl)

These volumes produce 50 µl PCR mix per tube.

Pipette mix 10 times at 32 μl stroke, and then briefly centrifuge.

Place on the preprogrammed thermal cycler and run the PCR program.

SAFE STOPPING POINT

If you are stopping, leave the samples on the thermal cycler at 4°C or store samples at 2°C to 8°C overnight.