Integrated Analysis of Single Cell Transcriptome and Surface Proteome

Integrated Analysis of Single Cell Transcriptome and Surface Proteome

The single cell surface proteome is a new technology for the detection of cell surface proteins at the level of single cells. CD Genomics offers a service based on 10X Genomics' single cell surface proteome technology, which can detect the number of single cell surface proteins and the expression of the corresponding single cell transcriptome.

How It Works

10X Genomics' single cell surface proteome is implemented by adding the detection of cell surface proteins to single cell transcriptome technology. This is achieved by tagging target proteins on the cell surface with specific antibodies linked to DNA tag chains, converting these DNA tags into sequencing results during library building and sequencing, and finally performing data analysis.

Gel beads used for single-cell surface proteomics are connected with two other tag chains in addition to standard tag chains compared with gel beads for single-cell transcriptome. Capture Seq 1 and Capture Seq 2 sequences were linked to the 3' ends of the two tag strands, respectively. The role of these two tag chains can be used to capture research targets that researchers are interested in, such as target proteins.

Using Chromium of 10X Genomics Company, the suspension dispersed into single cells and gel microbeads are mixed with the oil phase through microfluidics to form small water-in-oil droplets.

The Capture Seq 1 label on the beads binds to the label on the antibody bound to the cell surface. Next, under the action of polymerase, the tag chain is extended and connected to the Feature Barcode sequence on the antibody. Subsequent sequencing results including the three characteristic sequences of 10x Barcode, UMI, and Feature Barcode can be used as the original sequencing data for bioinformatics analysis.

Next, the cell membrane is broken, and the mRNA is released from the cell. The dissociated mRNA is mixed with the aqueous phase in the small droplet, that is, it contacts with the reverse transcriptase,the nucleic acid label strand primer bound on the gel beads, and the dNTP substrate in the aqueous phase. Next, a reverse transcription reaction occurs. The mRNA combines with the labeled DNA primer strand on the gel microbeads, and under the action of reverse transcriptase, cDNA is reverse transcribed.

All the aqueous phase of the emulsion is extracted, i.e. all the DNA strands with antibody tags and cDNA molecules with tags are extracted, and these cDNA molecules are added with adapters, amplified by PCR, and the illumina sequencing library is constructed and sequenced by the Illumina sequencer. After sequencing is completed, data analysis is performed.

Technical Highlights

1. The protocol is compatible with whole cells and nuclei

2. Compatible with targeted gene expression

3. Easy-to-use data analysis and visualization software

4. Adopt the dual index for library building strategy to maximize the reliability of sequencing results

For research use only, not intended for any clinical use.

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