Can I generate antibody data without evaluating gene expression with GEM-X Flex?

Question: Can I generate antibody data without evaluating gene expression with GEM-X Flex?

Answer: Yes, the GEM-X Flex protocol can be modified for customers that want to process up to 64 samples and 1.28 million cells for protein analysis without gene expression (“antibody-only”). This modified supported protocol can enable customers already performing flow cytometry or mass cytometry experiments to more easily increase the number of antibodies in their panel. 

To run the “antibody-only” workflow, customers will follow the GEM-X Flex Gene Expression Reagent Kit for Multiplex samples with Feature Barcode technology for Protein Expression (CG000789) with a few minor deviations highlighted below.

Kits Required:

Listed below are the GEM-X Flex part numbers that are required to run the antibody-only configuration. Please note, this configuration will not provide sufficient Flex Transcriptome Probes to enable Gene Expression analysis.

Overview of Kits:

• GEM-X Flex Sample Preparation v2 Kit (PN-1000781) for sample fixation
  • Please note: multiple kits may be required depending on how many unique samples are being processed and if intracellular protein (ICP) labeling is performed
  • A single GEM-X Flex Sample Preparation v2 Kit is sufficient for 24 rxn of ICP labeling and 48 rxn of cell surface protein labeling  
• GEM-X Flex Gene Expression Human/Mouse, 4 samples (PN-1000792/1000796) and GEM-X Flex Gene Expression Chip Kit, 4 chips (PN-1000791) will be sufficient to generate 4x GEM and library construction reactions
• Flex Feature Barcode Kit, 64 rxns (PN-1000628) and the GEM-X Flex Supplemental Wash Kit (PN-1000828) provide sufficient reagent for generating 4x 16-plex pools 
  • Please note: Conc. Post Hyb Buffer B (PN-2001308) will be used in place of Hyb Buffer B to set up 64x Barcode Oligo Hybridizations

Workflow Modifications:

To generate GEM-X Flex Protein Expression libraries without utilizing Gene Expression probes, the Hybridization step needs to be modified. After labeling with oligo-conjugated antibodies and fixation, samples are Hybridized with Antibody Multiplexing Barcodes. Workflow modifications are as follows (see page 52 of User Guide):

• Step 1.1b (pg 52): Do NOT prepare Hyb Mix. Instead, thaw Conc Post-Hyb Buffer B (PN-2001308) at room temperature, and prepare Post-Hyb Wash Buffer B
  • Vortex briefly and keep at room temperature; DO NOT keep at 4°C 

• Step 1.1c (pg 53): Incubate Post-Hyb Wash Buffer B at 37C in place of Hyb Mix
• Step 1.1d: Centrifuge fixed sample resuspended in Quenching Buffer B at 850 rcf for 5 min at 4°C
• Step 1.1e: Remove the supernatant
• Step 1.1f: Resuspend each pellet in 50 μl Post-Hyb Wash Buffer B
• Step 1.1g: Do NOT add 10 μl unique Human/Mouse WTA Probes BC001-BC016
• Step 1.1h: Add 2 μl of any Antibody Multiplexing Barcode (AB001-AB016) to each tube and gently pipette mix 10x with pipette set at 40 μl
  • Record Antibody Multiplexing Barcode name and PN used for each sample
  • Only one Antibody Multiplexing Barcode should be added to each tube
• Step 1.1i: Incubate sample for 1 hour at 37°C in a thermal cycler or a thermomixer with heated lid and no shaking 
  • Overnight hybridization is not required
• Proceed with Step 2: Post-Hybridization Washing & Pooling, following steps as instructed, but perform all wash steps at 37°C

Cell Calling:

Cell Ranger cell calls may need to be adjusted when analyzing antibody-only data. If so,  --force-cells can be used to adjust cells called. See this article for additional information.

Products: Flex Gene Expression