Index SNT Samples

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If a sample has been labeled with both Additive Primer A-compatible and Additive Primer H-compatible SNT labeling, perform the library preparation procedure individually for each type of tag (A or H). To enrich the appropriate synthetic tag library, use unique index combinations for each. You cannot perform the library preparation procedure for both types of tags within the same reaction.

• 4X PCR Master Mix (PMM4)
• Nuclease-free water
• P7 Index for use with Additive Primer A or H
• Universal P5 Index
1. PMM4—Pipette to mix, and then centrifuge briefly.
2. Save the following PCR program on the thermal cycler:
• Set the lid temperature to 105°C
• 95°C for 3 minutes
• X cycles of:
• 98°C for 20 seconds
• 60°C for 30 seconds
• 72°C for 20 seconds
• 72°C for 5 minutes
• Hold at 4°C

Adjust the program to optimize for different for sample and staining types.

Sample Input

Number of PCR Cycles (X)

Libraries compatible with Additive Primer H

7–10

Libraries compatible with Additive Primer A

12–15
1. Quantify 2 μl of each amplified sample using a Qubit High Sensitivity kit.
2. Prepare 10–15 ng of each purified SNT cDNA in separate wells of an 8-tube strip on ice.

If you cannot input 15 ng of SNT cDNA due to low yield, input half your elution volume (10 µl) and increase the PCR cycle number to at least 10. Save the other half of the elution volume at -20°C for cycle number troubleshooting if necessary.

3. Choose and record a unique index combination for each sample to make sure that no sample index combinations overlap in a multiplexed sequencing run (including whole transcriptome, A-compatible SNT libraries, and H-compatible SNT libraries).

If using the Illumina Single Cell Unique Dual Indexes when designing SNT indexes, do not use the same indexes provided in the Illumina Single Cell 3′ RNA kits. These are provided for the whole transcriptome libraries and contain a subset of indexes in the Illumina Single Cell Unique Dual Indexes plate, which can create indistinguishable final libraries during sequencing. Refer to Illumina Adapter Sequences for more information.

4. Combine the following volumes to prepare 50 µl total reaction volume:

Reagent

Volume Per Sample (μl)

10-15 ng purified synthetic tag sample

Variable

PMM4

12.5

P7 index for use with additive primer A or H 70X (10 µM)

2.5

Universal P5 index 50X (10 µM)

2.5

Nuclease-free water

Variable

The volume of nuclease-free water to add varies based on the volume of synthetic tag sample.

5. Pipette 10 times at the 40 μl stroke, and then centrifuge briefly.
6. Place on the preprogrammed thermal cycler and run the PCR program.

SAFE STOPPING POINT

If you are stopping, store samples overnight at 2°C to 8°C.