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Can GEM-X Flex customers perform TotalSeq™️-B Singleplex experiments?

Question: Can GEM-X Flex customers perform TotalSeq™️-B Singleplex experiments?

Answer: We highly encourage users to instead use Proteintech Genomics antibody panels or TotalSeq™️-C style conjugated antibodies following GEM-X Flex Gene Expression Reagent Kit for Singleplex samples with Feature Barcode technology for Protein Expression (CGCG000788) and purchase the Fixed RNA Feature Barcode Multiplexing Kit, 64 rxns (PN-1000628).

With that said, users can use TotalSeq™️-B antibodies with GEM-X Flex Sinlgeplex experiments with some workflow modifications. Users have two options for using TotalSeq™️-B antibodies with their Singleplex GEM-X experiments:

  1. Option One: Purchase the Fixed RNA Feature Barcode Kit, 16 rxns (PN-1000419) OR
  2. Option Two: Purchase the GEM-X Single Cell 3 Feature Barcode Kit v4, 16 rxns (PN-1000702) to acquire Amp Mix (PN 2000047) and purchase Truseq R2 primer from IDT

These modifications are discussed in detail below, however, please note these workflow modifications are not officially supported by 10x Genomics. We have developed additional resources to help enable these experiments to improve customer success.

 

Option One:

If the Fixed RNA Feature Barcode Kit (PN-1000419) has been purchased, users can utilize this kit with GEM-X Flex Singleplex experiments by performing modifications to our GEM-X Flex Gene Expression Reagent Kit for Singleplex samples with Feature Barcode technology for Protein Expression (CG000788), which can be reviewed below:

  1. Follow the Demonstrated Protocol for Cell Surface & Intracellular Protein Labeling for GEM-X Flex Gene Expression (CG000781) to label your cells with TotalSeq™️-B antibodies.
  2. Follow the GEM-X Flex Gene Expression Reagent Kit for Singleplex samples with Feature Barcode technology for Protein Expression (CG000788) User Guide:   
    1. Users will not need to purchase the Fixed RNA Feature Barcode Multiplexing Kit, 64 rxns (PN 1000628).  Therefore, on Step 1.1h on page 41, skip the addition of 2uL of Antibody multiplexing barcode oligo to the probe hybridization reaction.
    2. On Step 4.2a on page 63, replace Pre-Amp Primers C (PN-2000953) with Feature Pre-Amp Primers (PN-2000515), supplied in the Fixed RNA Feature Barcode Kit (PN-1000419). 
      • This pre-amplification PCR will amplify both the GEX product (Truseq R1/smRNA R2) and Protein product (Truseq R1/Truseq R2).
    3. On Step 6.1 on page 73, follow this section to make your TotalSeq™️-B protein library. On Step 6.1 f. replace the Dual Index Plate TN Set A (PN-3000510) with Dual Index Plate TT Set A (PN-3000431).

Option Two:

If users have NOT purchased the Fixed Feature Barcode Kit (PN-1000419), TotalSeq™️-B experiments with GEM-X Flex can still be performed. Users will need to instead purchase the GEM-X Single Cell 3 Feature Barcode Kit v4, 16 rxns (PN-1000702; 1 tube of Amp Mix) or 3’ Feature Barcode Kit, 16 rxns (PN-1000262; 3 tubes of Amp Mix) to acquire Amp Mix (PN 2000047) and purchase a 25nmol, standard desalted Truseq R2 primer from IDT (normalized to 50μM) with the following sequence: 5’-GTGACTGGAGTTCAGACGTGTG-3’ and perform the following modifications:

  1. Follow the Cell Surface & Intracellular Protein Labeling for GEM-X Flex Gene Expression (CG000781) to label your cells with TotalSeq™️-B antibodies.
  2. Follow the GEM-X Flex Gene Expression Reagent Kit for Singleplex samples with Feature Barcode technology for Protein Expression (CG000788) User Guide:
    1. Users will not need to purchase the Fixed RNA Feature Barcode Multiplexing Kit, 64 rxns (PN 1000628).  Therefore, on Step 1.1h on page 41, skip the addition of 2uL of Antibody multiplexing barcode oligo to the probe hybridization reaction.
    2. On Step 4.2a on page 63, add 2μL of the TruseqR2 primer (50µM) to the 35μL of the pre-amplification mix (25μL Amp Mix (PN-2000047), 10μL Feature Pre-Amp Primers (PN-2000515)). 
      • This pre-amplification PCR will amplify both the GEX product (Truseq R1/smRNA R2) and Protein product (Truseq R1/Truseq R2).
    3. On Step 6.1 on page 73, follow this section to make your TotalSeq™️B protein library).  On Step 6.1 f. replace the Dual Index Plate TN Set A (PN-3000510) with Dual Index Plate TT Set A (PN-3000431).

Note, in either case, for Cell Ranger data analysis, the pipeline can be run without any deviations.

Product: Single Cell Gene Expression Flex, Flex Gene Expression