What is the importance of emulsion-safe plastic consumables?

Question: What is the importance of emulsion-safe plastic consumables?

Answer: Some plastics can interact with and destabilize GEMs. It is therefore critical to use validated emulsion-safe plastic consumables when handling GEMs. This article lists the pipette tips and PCR strip tubes that have been validated for 10x Genomics Single Cell assays, and provides examples of the potential impact on data quality if non-validated plastics are used. 

Impact on data quality

Plastics other than those recommended here may interact with and destabilize GEMs. Alternative plastics may result in the premature breaking of GEMs before or during the GEM-RT/barcoding reaction, allowing the contents of individual cells and barcoded primers from individual Gel Beads to pool. This will result in irreparable loss of single cell resolution. Alternative plastics may also negatively impact the performance of the GEM incubation reaction resulting in low yield after amplification.

Plastics that may impact emulsion stability include brands from Bio-Rad, Denville, Axygen, and BioPlastics. The image below shows GEMs that were transferred to Denville branded tubes. The image was taken immediately after RT. Axygen tubes saw similar deterioration in emulsion stability after 24 hours, although nothing visually notable after RT or up to 12 hours.   

Using plastics that impact emulsion stability may compromise the single cell nature of the data. For example, using unsupported PCR strip tubes (BioPlastics) for the GEM-RT step may lead to a “second knee” in the Barcode Rank Plot, due to premature breaking of GEMs.

Validated plastics for GEM-RT

We have validated the following strip tubes in all manual single cell assays:

• TempAssure PCR 8-tube strip (USA Scientific) - Part Number (US) 1402-4700
• PCR Tubes 0.2 ml 8-tube strips (Eppendorf) - Part Number (US) 951010022 or Fisher E0030124286
• MicroAmp 8 Tube Strip (Thermo Fisher) - Part Number (US) N8010580 (tubes) and N8010535 (caps)

The PCR Tubes from Eppendorf are available for order in Europe - Part Number 0030124359.

For automated Single Cell 3' and Single Cell 5' VDJ applications, the Eppendorf twin.tec 96-well PCR plates are recommended (see What consumables are supported for use with the Chromium Connect Instrument?).

Validated pipette tips

Rainin pipette tips have been extensively validated by 10x Genomics and are highly recommended for all single-cell assays. If Rainin tips are unavailable, any of the listed alternate pipette tips validated by 10x Genomics may be used. See Technical Note CG000492. 

*Eppendorf pipette tips require Eppendorf pipettors. All other tips are compatible with Rainin pipettors.

**These VWR tips have been discontinued.

For steps downstream of the GEM-RT incubation step, it may be possible to use pipette tips from other vendors if Rainin tips or the alternate tips are in limited supply. We anticipate that any other pipette tips suitable for molecular biology assays would be compatible with downstream steps in 10x assays.

Therefore if you have limited access to Rainin tips, we would recommend saving/conserving your Rainin tips and using them only for steps involving handling GEMs. Tips from other vendors may be used for downstream steps in the assay.

We have not tested alternative tips for the Visium workflows. However, it is low risk that application performance is impacted since no emulsion handling or microfluidics are involved.

Products: Single Cell Gene Expression, Single Cell Immune Profiling, Single Cell ATAC, Single Cell Multiome ATAC+GEX, Fixed RNA Profiling Gene Expression, Single Cell Gene Expression Flex