For this collaboration, Immunai will perform and fund scRNA sequencing of patient samples, covering the whole transcriptome, surface proteome (80+ surface proteins), and TCR repertoire. Immunai will supply raw scRNA sequencing data, a QC report, and cell annotations to the research center.

A: For PBMCs we ask for a minimum of 500K viable cells.
B. Tissues can be either:

• Fresh samples stored in a cryopreservation medium. If the tissue is larger than 8mm (e.g. tumor resections), ideally shortly after excision, it should be cut in smaller fragments (see below).
• Formalin fixed samples embedded in paraffin and provided as blocks/curls

PBMCs for scRNAseq/CITEseq – Whole blood can be collected using any of the following anticoagulant vacutainer tubes: Sodium Heparin, Sodium Citrate, K2EDTA according to the manufacturer instructions. PBMCs need to be isolated using a Ficoll gradient within 2-4 hours of collection to maintain PBMC viability. After PBMC isolation, we recommend cells be eluted in FBS + 10% DMSO (or equivalent) freezing media into cryovials compatible with LN2 vapor-phase storage conditions. Samples should be controlled-rate frozen using a device that cools samples at -1°C per minute (ex. Nalgene® Mr. FrostyTM ). Long-term cell storage temperature is recommended to be -150°C or colder.

Fresh Tissue for scRNAseq/CITEseq–  Ideally, upon excision, the tumor is immediately cut in smaller fragments (unless it’s a small, <8mm, biopsy). If that is not possible, it can be collected in MACS tissue storage solution and kept on ice for a few hours until it’s ready for cutting. Long term storage at this stage is not recommended. Tissue fragments (up to 16) are placed  in a cryotube containing 1 mL CS10,  making sure all fragments are well submerged. Samples should be controlled-rate frozen using a device that cools samples at -1°C per minute (ex. Nalgene ® Mr. Frosty TM ). Long-term cell storage temperature is recommended to be -150°C or colder.

FFPE tissue for scRNAseq or in situ transcriptomics – Fix tissue  in 10% Neutral Buffered Formalin or 4% formaldehyde at a fixative to tissue ratio of 20:1 for optimal performance. Fix at 4°C for ~16-24 h to ensure proper fixative penetration. Dehydrate in an ethanol series to displace any water remaining in the tissue. Clear tissue using a solvent to remove any remaining ethanol. Apply wax to the tissue and allow it to infiltrate. Place the infiltrated tissue in a mold, and add more melted paraffin to surround and encase the tissue. For optimal storage, store at 4°C and avoid exposure to high temperatures or humidity.

Immunai will cover the sequencing costs for this collaboration. All other expenses, including sample collection, data collection, regulatory fees, shipping, and insurance, will be the responsibility of the research institute.

Immunai’s primary focus is on deciphering the immune system. Therefore, any research question that contributes to a better understanding of the human immune system will be considered.

The timeline for this collaboration is flexible and will be determined by the size of the cohort and Immunai’s internal resource availability. Specific timelines will be mutually discussed and agreed upon with each collaborator.

Immunai prioritizes projects with sufficient samples to yield significant scientific results, aligning with the collaboration’s goal of advancing scientific knowledge.

Immunai upholds the highest standards, ensuring all studies comply with regulatory and legal requirements and possess the necessary approvals. In line with the collaboration’s goals, this collaboration is open to non-commercial academic research only.