Tips and Techniques

Working with RNA

Before executing this protocol, become familiar with working with RNA. The following section provides general guidelines for working with RNA, but the guidelines are not all-inclusive.

•

Due to the ubiquitous presence of RNases, RNA is susceptible to degradation. RNases are robust nucleases specific to RNA. Unlike DNases, they are not easily denatured or inactivated.

•

Maintain reagents, pipettes, and a work space specifically for working with RNA. An enclosed space such as a PCR hood is recommended. Wipe down pipettes and the working space regularly with a laboratory alcohol cleaning solution (70% alcohol). Periodically (one time a week to one time a month), clean surfaces with a 10% sodium hypochlorite solution. Alternatively, commercial solutions are available to inactivate RNases.

•

Only use consumables and reagents that are nuclease-free and dedicated for RNA use. Handle reagents carefully to avoid RNase contamination.

•

The most common external RNase contamination comes from the skin. Handle any item used for RNA work with gloves to maintain nuclease-free surfaces. Wear a clean laboratory coat or gown to prevent shedding of skin or hair in and around the RNA work surface.

•

Dust and bacteria are also sources of RNases. Keep surfaces and items free of dust and work with RNA away from bacterial processes.

•

Treat refrigerator and freezer handles as RNase-free surfaces and only open them with gloved hands.

•

Exercise all standard laboratory best practices such as always wearing PPE, not touching rims of open tubes, and not reusing pipette tips. Seal consumable bags and reagent tubes tightly when not in use.

Handling Beads

•

Do not overdry beads. Proceed to the next step when the magnetic beads have air dried to the point of only a slight gloss remaining, but before they show cracks.

Working with PCR Products

Before executing this protocol, become familiar with the process of unidirectional PCR workflow and managing PCR amplicon products. Complete all steps in the Illumina Single Cell 3′ RNA workflow in a pre-PCR workspace until after the thermal cycling step during Amplify Library. Complete all remaining steps in the post-PCR workspace. One exception is the magnetic bead purification following Assess cDNA Quality, which also must be completed in the post-PCR workspace. The following section provides general guidelines for working with PCR products, but the guidelines are not all-inclusive.

•

PCR by design creates many amplicons, copies of the input material. This strength of PCR also comes with the risk of amplicon contamination of workspaces and materials.

•

It is best practice to maintain two separate workspaces for pre and post-PCR work. Ideally these spaces are physically divided and each have a dedicated set of equipment and materials. All work before a PCR amplification is to be performed in the pre-PCR workspace. The sample is then transferred to the post-PCR workspace for further processing. No material from the post-PCR workspace should ever come back into the pre-PCR workspace.

•

For some reagents and consumables, multiple sets may be required for pre-PCR and post-PCR workspace use to avoid carryover contamination.

•

If it is not possible to maintain two separate workspaces for pre and post-PCR work, it is still highly recommended to keep two separate sets of consumable materials and to maintain strict workplace sterility. Make sure all benches and equipment are regularly cleaned with a bleach solution or a commercial DNA degrader to reduce contaminating amplicons and always wear clean gloves.

Centrifuge Steps

•

Complete all centrifuge steps in the Illumina Single Cell 3′ RNA workflow after sample preparation where the sample is in 1.5 ml or PCR tubes with a benchtop minifuge using speeds of 2000 × g. For steps where the sample is in a 5 ml or 15 ml conical, a swinging bucket rotor centrifuge must be used with a speed of 750 × g.

•

Always use the power button to turn the benchtop minifuge off for a gradual stop to maintain well packed beads. Do not apply standard brakes, like opening the lid.

Thermal Cycler Lid Pressure

Follow the manufacturer's instructions for using PCR tube strips with your brand of thermal cycler. Thermal cyclers with a lot of lid pressure can cause the 8-tube strip to collapse during PCR amplification steps when only one is used. Use empty tube strips as braces surrounding both sides of the 8-tube strip to help balance the lid pressure for brands that do not include plastic frame supports.

PCR 8-Tube Strip Lids

When vortexing 8-tube strips, apply pressure to the lid to maintain a tight seal. Replace the lids on the 8-tube strip after each dry bath or thermal cycler program using the 105°C lid setting to prevent lid damage.

Cell Loading

This protocol describes addition of 200,000 cells into each reaction, resulting in recovery of > 100,000 cells and a multiplet rate of < 8%. The optimal input cell concentration is 10,000 live cells/µl when adding 20 µl cells plus 160 units RNase inhibitor into each PIP tube. If it is not possible to concentrate cells to 10,000 cells/µl, a larger volume can be input as long as the total loading volume including RNase inhibitor is ≤ 30 µl. It is important to note that increased cell input increases the observed multiplet rate.