This is a generic project plan for performing immune repertoire experiment from the management of the sample to the analyses of RepSeq data. Such experiment may comprise samples from a setting such as healty vs disease, vaccinated vs placebo, genetically modified organism vs. wild type, different tissues or time points… The aim of this project is to study immune repertoire wide differences between the samples on the level of V, J, CDR3, specificities and more…

STEP 1: SAMPLE PREPARATION

Pre-analytical management of your sample are critical for the best output of the experiment. At PAREAN biotechnologies, we work in just-in-time model to perform cell biology on fresh sample. After receiving your samples, our team carefully identified and prepares them for cell sorting experiment to manage them in the minimum of time.

We create a single-cell suspension and with FACS BD Melody, we sort the cell of interest from your sample. According to their phenotype and markers, T cells, B cells, NK T cells, naïve or activated, cytotoxic or regulatory … , all kind of cells could be sorted to investigate the immune repertoire. Cell can also be sorted based on their affinity for specific antigen (tumor associated antigen, viral antigen, autoimmune disease associated antigens…) to decipher specifically the pathology related immune repertoire. Cells are then lysed and RNA are perfectly conserved at -80°C in lysis buffer.

STEP 2: SAMPLE PROCESSING AND LIBRARY PREP

The protocol begins with RNA purification. You can send us isolated RNA, or sort your cells in any RNA lysis buffer compatible with NGS sequencing, before sending them to us. We perform RNA purification in our lab with your specificities. Quality control of RNA quality and quantity are performed by automated electrophoresis. According to RNA integrity, we choose the right method to amplify your sample and create cDNA library ready to sequence. Quality control of the cDNA quality and quantity are also performed by automated electrophoresis to ensure the good outcome of the sequencing. Once all these steps are performed, we complete the NGS library preparation and send the sample out for sequencing.

STEP 3: SEQUENCING AND DATA ANALYSIS

NGS libraries are sequenced function of the needed sequencing length and depth, for a cost-effective sequencing.

When we receive the raw sequencing data, we map it to the gold standard reference database of immune repertoire analysis (IMGT). We perform a QC analysis, a preliminary clustering and differential gene expression analysis.

For single cell repertoire analysis, mapping of sequencing data and initial quality control reports are done with the standard 10x Genomics Cell Ranger pipeline. You’ll be provided with a cloupe data file for browsing the data with the 10x Genomics Loupe Browser.

We’ll also provide you with a preliminary clustering and differential gene expression analysis, done with a customized version of the Seurat analysis pipeline.

The sequenced receptors are mapped to the antigens they bind to, revealing which diseases a patient’s immune system has seen or is actively fighting.

The two chains from each of the hundreds of thousands of T- or B-cell receptors can be paired back together in parallel to be used for therapeutic purposes.

Characterize Finally, we characterize them to learn which T-Cell Receptors may be most effective for cellular therapies or which B-Cell Receptors produce the most effective antibodies to fight or neutralize disease.