Synthesize cDNA

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• RTAM (RT Additive Mix)
• RTEM (RT Enzyme Mix)
• TSO1 (Template-Switch Oligo)
1. Prepare the following consumables:
• RTAM—Pipette to mix.
• RTEM—Pipette to mix.
2. Select Prog.C and then manually set the lid temperature to 105°C on the dry bath.

Refer to Dry Bath Operation to review program parameters.

3. Select Run, and then select Lid On to preheat the dry bath to 25°C for the reverse transcription program.

The dry bath steps specified in this protocol are specific to the PIPseq dry bath sold as part of the Illumina Single Cell Prep Starter Equipment Kit. If you are using a user-supplied Eppendorf dry bath, you must manually program the protocols. Refer to Eppendorf's documentation for more detailed information on how to operate their products.

1. Combine the following volumes to create RT Master Mix.

Reagent

Volume Per Reaction (μl)

4.4X Reaction Master Mix (µl)

8.8X Reaction Master Mix (µl)

RTAM

80

352

704

TSO1

8

35

70

RTEM

12

53

106

Total

100

440

880
2. Pipette to mix.
3. Centrifuge briefly to bring liquid to the bottom of the tube.
4. Add 100 μl RT Master Mix to each PIP tube.
5. Flick to disperse the pellet.
6. Pulse vortex for 5 seconds to mix, and then centrifuge for < 1 second.

If PIPs are pelleted in the centrifuge, repeat vortexing and centrifuge steps.

7. Place on the preheated dry bath. Select Skip, and then select Yes to run the cDNA Synthesis program, starting with incubation at 25°C for 30 minutes.

SAFE STOPPING POINT

If you are stopping, leave the samples in the dry bath at 4°C or store samples at 2°C to 8°C overnight.