Tech Sharing Starter Kits

One of our goals at the Center for Integrated Cellular Analysis is to distribute the innovative technologies we develop and enable the community to use them in their own research.

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.

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.

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

Tech Sharing kit interest form

If you are interested in receiving a kit, please fill out this form. We’ll follow up by e-mail with next steps and a request for a FedEx account number for shipping.

Retired Kits:

We previously introduced Cas13 RNA Perturb-seq (CaRPool-seq) which leverages the RNA-targeting CRISPR/Cas13d system and enables efficient combinatorial perturbations alongside multimodal single-cell profiling. CaRPool-seq encodes multiple perturbations on a cleavable array which is associated with a detectable barcode sequence, allowing for the simultaneous targeting of multiple genes.

For more details on the technology, including barcode, guide, and primer sequences, please check out https://carpool-seq.com/, as well as the CaRPool-seq manuscript

In 2022, we introduced nanobody-tethered transposition followed by sequencing (NTT-seq), a new assay capable of measuring the genome-wide presence of multiple histone modifications and protein-DNA binding sites at single-cell resolution. NTT-seq utilizes recombinant Tn5 transposase fused to a set of secondary nanobodies (nb). Each nb-Tn5 fusion protein specifically binds to different immunoglobulin-G antibodies, enabling a mixture of primary antibodies binding different epitopes to be used in a single experiment. You can read more about the technology in this July, 2022 genomeweb article.

For more details on the technology, please check out the NTT-seq manuscript or visit NTT-seq.com