Question: What sequencing configuration is required for Proteintech Group (PTG)-derived antibody libraries generated from multiplexing experiments using Flex Gene Expression, so that the data can be analyzed properly using Cell Ranger?
Answer:
To use Cell Ranger v8.0 and later to analyze multiplexed protein expression data generated using PTG antibodies, you can use the standard sequencing configuration as listed in the “Sequencing Parameters” section of the Multiplexed User Guide.
Standard Sequencing Configuration:
Read 1: 28 cycles
i7 index: 10 cycles
i5 index: 10 cycles
Read 2: 90 cycles
If using Cell Ranger v7.2, you will need to sequence both the multiplexed Gene Expression (GEX) and Protein Expression (Ab) library with an alternative sequencing configuration. This configuration is found in the “Alternate Sequencing Parameters” section of the Multiplexed User Guide. This configuration will be required for Cell Ranger analyses of multiplexed Ab-only libraries (pools not containing GEX libraries).
Alternative Sequencing Configuration:
Read 1: 48 cycles
i7 index: 10 cycles
i5 index: 10 cycles
Read 2: 50 cycles
This read scheme, which we refer to as MFRP-R1 chemistry, allows both the GEX Probe and Antibody Multiplexing Barcode to be extracted in the same positions of Read 1. The alignment of the Multiplexing Barcodes is required by Cell Ranger to properly demultiplex sample barcodes. Please note that for Singleplex GEX and Ab libraries, there is no need to utilize the GEX Probe Barcodes and Antibody Multiplexing Barcodes during analysis, so the default sequencing configuration of Read 1 (28 cycles), i7 index (10 cycles), i5 index (10 cycles), and Read 2 (90 cycles) can be used.
In the Alternate Sequencing Configuration, the following information is extracted from Read 1 for both the GEX and Ab library types:
Cycles 1 - 16: GEM barcode
Cycles 17 - 28: UMI
Cycles 29 - 40: pCS1 constant sequence (discarded)
Cycles 41 - 48: GEX Probe and Ab Multiplexing Barcodes
This read configuration also requires a 10% PhiX spike-in when sequencing on the NovaSeq 6000 or a 5% PhiX spike-in when sequencing on the MiSeq, NextSeq 500/550, or NextSeq 2000.
If interested in sequencing antibody libraries derived from using Proteintech Group (PTG) antibodies mixed with Biolegend antibodies please see the following article: What sequencing configuration is required for antibody libraries derived from using Proteintech Group (PTG) antibodies mixed with Biolegend antibodies, generated from multiplexing experiments using Single Cell Gene Expression Flex?
For details on how to perform intracellular protein labeling, see the following article: Is the Fixed RNA Profiling assay compatible with intracellular protein labeling?
Products: Flex Gene Expression