||Price per sample
||Low depth: average 100 000 sequencing reads per each TCR alpha and TCR beta library
||Medium depth: average 500 000 sequencing reads per each TCR alpha and TCR beta library
||Deep depth: average 2 000 000 sequencing reads per each TCR alpha and TCR beta library
** Please note that minimal order is 12 samples.
- RNA extraction from RLT® buffer
- TCR alpha and beta cDNA libraries generation using 5’RACE with unique molecular identifiers (UMI)
- Sequencing on MiSeq®, HiSeq®, or NextSeq® 150+150 nt paired end mode
- UMI-based grouping of reads with error correction using MIGEC™ software
- Extraction of TCR alpha and beta repertoires using MiXCR™ software, with additional error correction and filtering of non-coding CDR3 variants
- Basic bioinformatics analysis of each TCR alpha and beta repertoire characteristics: number of TCR cDNA molecules and extracted CDR3 clonotypes; average CDR3 and NDN length; average physicochemical characteristics of amino-acid composition in the middle of CDR3; normalized diversity metrics
Results delivery includes:
- Raw sequencing data in FASTQ format
- Report with sequence quality controls
- Extracted TCR alpha and beta CDR3 repertoires on each sample
- A single table with basic characteristics for each repertoire
Advanced bioinformatics analysis is available on request, not included in service price.
We accept RNA isolated by any standard method in GenTegra/RNAstable tubes, or cell lysate in RLT® buffer (QIAGEN®) in 1.5 ml screw-cap conical microcentrifuge tubes sealed with parafilm, prepared one of the following ways:
- At least 100 live T cells sorted/placed directly in 50-500 ul of RLT® buffer. The volume of RLT® buffer should not be diluted more than 20% during sorting. 50,000 sorted cells may carry the volume around 50-80 ul when using 70 um nozzle for cell sorting. The cells are lysed immediately in the collection tube and mRNA is protected from degradation. Lysed cells can be stored in RLT® buffer at -70°C for at least 6 months.
- At least 10 ng of high quality RNA isolated from T cells or PBMC or from T cell-containing tissue. QIAGEN® kits recommended for simplicity, skilled TRIzol® isolation for maximal yield.
- Single cell suspension properly prepared from fresh T cell-containing tissue sample, placed in RLT® buffer.
Please note that RNA quality is critical for our pipeline. The choice of the appropropriate protocol for RNA extraction depends on the amount of starting material.
RNA quality control tests (QC)
Our quality control test includes cDNA synthesis and amplification with further analysis of obtained PCR product.