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How to use custom V(D)J primers on Chromium Connect?

Question: How to use custom V(D)J primers on Chromium Connect?

Answer: The Single Cell 5’ V(D)J workflow on Chromium Connect supports human and mouse TCR/BCR library construction. However, the use of custom primers for non-human and non-mouse species requires careful preparation in order to be used on the Chromium Connect platform. 

While no impact on assay performance is anticipated, the use of custom primers in the automated assay is not supported or validated by 10x Genomics. 10x Genomics cannot guarantee that customer primers will yield data comparable to the data showcased in the Chromium Connect: Consistent Automated Single Cell 5' Gene Expression and V(D)J Library Generation technical note. 

Follow the steps below to enable the use of custom primers in the Automated V(D)J workflow:

  1. Design primer mix 1 and primer mix 2 for the species of interest. See this Knowledgebase Article for recommendations on primer design, quality control, and concentration optimization. Notably, automated and manual workflows share the same master mix recipe for V(D)J amplifications. Therefore, primer concentration guidelines for the manual workflow can be directly applied to the automated workflow. See details of the master mix breakdown in this User Guide.
  2. Once the primers have been optimized, load 850 μl of primer mix 1 into a 2 mL tube (Pictured below is Thermo Fisher Scientific 3488NK). A comprehensive list of alternative 2 mL tubes can be found in this Knowledgebase Article.
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  3. Load 170 μL of primer mix 2 into a 0.5 mL tube (Pictured below is Thermo Fisher Scientific 3472NK). A comprehensive list of alternative 0.5 mL tubes can be found in this Knowledgebase Article.

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  1. Vortex both primer tubes for 10 seconds at max speed. Centrifuge briefly.
  2. Initiate the V(D)J workflow on the Chromium Connect User Interface. Leave “Species-Cell Type?” selection as the default value: Human-TCR.
  3. Proceed to prepare other reagents and load the deck following instructions displayed on the screen.
  1. Instead of loading Human or Mouse T/B Cell Primer Mixes, load custom primer mix 1 to position P1 and custom primer mix 2 to position P2 on carrier 3. Carefully crosscheck the loading positions with the schematics on the user interface. 


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  1. Proceed to scan reagents and consumables on Chromium Connect. Since the two custom primer mixes have no barcodes, the scanning result will show as “Not Read.”
  2. Follow the instructions on the screen to start the assay.

After assay completion, follow the onscreen prompts to cleanup the instrument deck and move forward with collecting the final product, sequencing, and conducting data analysis. If running the Cell Ranger or Cell Ranger VDJ pipelines with custom primers, refer to this on detecting gamma-delta chains in V(D)J data. 

 

Product: Chromium Connect