*Please scroll down to the bottom of the page for the final deliverable.
Antibody-oligonucleotide conjugates are required in a number of expanding applications, particularly in single cell multiomics where extensive data sets can be generated at the molecular level from a single cell. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) combines scRNA-Seq and Proteomics in one application, to quantify RNA and protein at the same time. This page explores the capabilities of custom antibody-oligo conjugations, and their applications.
TotalSeq™ reagents are designed to seamlessly integrate into the CITE-seq workflow developed by Stoeckius, et al.
But they are compatible with most droplet-based single cell sequencing methods including the 10x Genomics platform or Drop-seq. They are also compatible with the similar workflow developed by Peterson, et al. In termed, REAP-seq.
Prior to droplet separation and sequencing, cells are incubated with TotalSeq antibodies and unbound antibody washed away.
The cells then undergo the typical single cell RNA-seq protocol via droplet separation.
And then reverse transcription to obtain cDNA.
Followed by cDNA amplification.
After amplification, the total cDNA mix is separated into the Antibody-Derived Tags or ADTs, generated from the the TotalSeq™ conjugates and the mRNA-derived cDNA so that the sequencing libraries can be prepared independently.
Preparing separate TotalSeq ADT and mRNA sequencing libraries is critical for ensuring reliable and balanced proteomics and genomics data.
With a small size of only 180 base pairs in length, the TotalSeq™ barcodes are easily size separated from mRNA-derived cDNA.
Once the two libraries are prepared, they can be pooled at the ratio needed to achieve an optimal read depth for each library.
Thus, both proteomics and transcriptomics information can be obtained from a single sequencing run.
Upon completion of NGS next generation sequencing,
The identity and abundance of proteins is reflected by the number of reads corresponding to each TotalSeq barcode as part of a standard bioinformatics analysis pipeline.
Get started with TotalSeq™—find out which antibody-oligo conjugates are available by visiting Biolegend.com/totalseq.