CICA Tech Sharing: Phospho-seq
Click here for Phospho-seq Protocols
On the Phospho-seq website you’ll find additional information on protocols, reagents, sequencing, computational tools, and FAQ.
Each Phospho-seq Starter Kit contains the following reagents:
5 ul 20 mM mTz-PEG4-NHS (Click Chemistry Tools: 1069-10)
One 50 kDa MW cutoff filter (EMD Millipore: UFC 505024)
Four 30 kDA MW cutoff filters (EMD Millipore: UFC 503096)
20 ul 1 M glycine (BioBasic – GB0235-500 (powder))
15 ml 1X BBS buffer (Rockland - MB-050-1000)
50 ul TCO-PEG4-Gly (Made from Click Chemistry Tools: A137-10 and glycine)
TCO-PEG labeled TSB oligos
10 ul of 4 separately indexed oligos
5 ul (3 ug) Pre-conjugated pRPS6 antibody
5 ul Bridge Oligo B
Protocol
The protocol is detailed in the Phospho-seq manuscript and a full step-by-step protocol is available on Phospho-seq.com
The starter kit includes materials to conjugate up to four antibodies of the user’s choice with four separately indexed labeled TSB DNA oligos as well as a pre-conjugated control antibody for phospho-RPS6. We also provide a bridge oligo for use in the 10X scATAC-seq and scMultiome kits for antibody capture.
In 2023 we introduced Phospho-seq, an integrated approach that aims to quantify phosphorylated intracellular and intranuclear proteins, and to connect their activity with cis-regulatory elements and transcriptional targets. We utilize a simplified benchtop antibody conjugation method to create large custom antibody panels for simultaneous protein and scATAC-seq profiling on whole cells
We have observed that one of the largest obstacles in performing antibody-based single-cell protein profiling including ASAP-Seq, InCite-Seq, NEAT-Seq, QURIE-Seq and others is the limited commercial availability of pre-conjugated antibodies, especially for intracellular proteins. For Phospho-seq we adapted an already established click-chemistry-based DNA-antibody conjugation method to create large panels of custom conjugated antibodies.
We hope that these kits remove some of the barriers of entry to using this technology and are excited to see what users do with them!