Synthesize First Strand cDNA

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This step reverse transcribes the hexamer-primed RNA fragments to produce first strand cDNA. The First Strand Synthesis Mix includes Actinomycin D, which allows RNA‑dependent synthesis and improves strand specificity while preventing spurious DNA‑dependent synthesis.

• FSA (First Strand Synthesis Mix)
• RVT (Reverse Transcriptase)
• 1.7 ml microcentrifuge tube, RNase-free
• Microseal 'B' adhesive film
• Prepare for a later procedure:
– RSB (Resuspension Buffer)
– SMM (Second Strand Marking Master Mix)
– AMPure XP (Agencourt AMPure XP)

This set of reagents contains potentially hazardous chemicals. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Wear protective equipment, including eye protection, gloves, and laboratory coat appropriate for risk of exposure. Handle used reagents as chemical waste and discard in accordance with applicable regional, national, and local laws and regulations. For additional environmental, health, and safety information, refer to the SDS at support.illumina.com/sds.html.

1. Prepare the following consumables:

Reagent

Storage

Instructions

RSB

-25°C to -15°C

Thaw at room temperature. Vortex and invert to mix.

RVT

-25°C to -15°C

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

SMM

-25°C to -15°C

Thaw on ice. Invert to mix, and then centrifuge briefly.

AMPure XP

2°C to 8°C

Let stand for 30 minutes to bring to room temperature. Vortex and invert to mix.
2. Save the following FSS program on the thermal cycler:
• Choose the preheat lid option and set to 100°C
• Reaction volume is 25 µl
• 25°C for 10 minutes
• 42°C for 15 minutes
• 70°C for 15 minutes
• Hold at 4°C
1. In a 1.7 ml tube on ice, combine the following volumes to prepare First Strand Synthesis Master Mix. Multiply each volume by the number of samples.
• FSA (9 µl)
• RVT (1 µl)

Volumes include reagent overage for accurate pipetting.

2. Thoroughly pipette First Strand Synthesis Master Mix to mix.
3. Centrifuge the sealed PCR plate at 280 × g for 10 seconds.
4. Add 8 µl First Strand Synthesis Master Mix to each well.
5. Pipette 10 times, and then seal.
6. Place on the preprogrammed thermal cycler and run the FSS program.

Total program time is ~43 minutes and each well contains a volume of 25 µl.