Synthesize Second Strand cDNA

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This step removes the RNA template and synthesizes a replacement strand to generate blunt-ended, double-stranded cDNA fragments. Magnetic beads then separate the cDNA from the Second Strand Synthesis Master Mix.

• IPB (Illumina Purification Beads)
• RSB (Resuspension Buffer)
• SMM (Second Strand Marking Master Mix)
• 96-well PCR plate, semiskirted
• Freshly prepared 80% ethanol (EtOH)
• Microseal 'B' adhesive film
• IPB
– Use at room temperature.
– Resuspend before each use.

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:
• RSB—Vortex and invert to mix.
• SMM—Invert to mix, and then centrifuge briefly.
2. Prepare 80% EtOH from absolute EtOH.
3. Save the following SSS program on the thermal cycler:
• Choose the preheat lid option and set to 40°C
• Set the reaction volume to 50 µl
• 16°C for 1 hour
• Hold at 4°C

Total program time is ~1 hour.

Generate cDNA

1. Centrifuge the PCR plate at 280 × g for 10 seconds.
2. Invert SMM to mix, and then centrifuge briefly.
3. Add 25 µl SMM to each well.
4. Pipette 10 times, and then seal.
5. Centrifuge at 280 × g for 10 seconds.
6. Place on the preprogrammed thermal cycler and run the SSS program.

Each well contains a volume of 50 µl.

Clean Up cDNA

1. Centrifuge the PCR plate at 280 × g for 10 seconds.
2. Resuspend IPB as follows.
a. To mix, invert the bottle manually for 2 minutes, at a rate of 1 inversion per second. While inverting, rotate the bottle 90 degrees every 30 seconds.
b. If beads are still adhered to the walls of the container, invert the bottle manually for an additional 1 minute.
3. Add 90 µl IPB to each well.
4. Seal and shake at 2200 rpm for 1 minute.
5. Incubate at room temperature for 5 minutes.
6. Centrifuge at 280 × g for 10 seconds, and then unseal.
7. Place on a magnetic stand and wait until the liquid is clear (~5 minutes).
8. Remove and discard all supernatant.
9. Wash beads as follows.
a. Keep on the magnetic stand and add 175 µl fresh 80% EtOH to each well.
b. Wait 30 seconds.
c. Without disturbing the beads, remove and discard all supernatant.
10. Wash beads a second time.
11. Using a 10 µl pipette, remove and discard all residual EtOH.
12. Air-dry on the magnetic stand for 2 minutes. Do not overdry the beads.
13. Remove from the magnetic stand.
14. Add 19.5 µl RSB to each well.
15. Seal and shake at 2700 rpm for 1 minute.
16. Incubate at room temperature for 2 minutes.
17. Centrifuge at 280 × g for 10 seconds, and then unseal.
18. Place on the magnetic stand and wait until the liquid is clear (~4 minutes).
19. Transfer 17.5 µl supernatant from each well to a new PCR plate.

Small amounts of bead carryover do not affect performance.

SAFE STOPPING POINT

If you are stopping, seal the plate and store at ‑25°C to ‑15°C for up to 7 days.