RNA isolation and cDNA synthesis from CSF cell pellets were performed using the μMACS One-step cDNA Kit (Miltenyi Biotec) according to the manufacturer’s instructions. Synthesised cDNA was eluted from the column and collected with 50 µL cDNA elution buffer. Immunoglobulin Kappa Variable (IGKV) and Immunoglobulin Lambda Variable (IGLV) sequencing of cDNA were performed by Adaptive Biotechnologies (Seattle, Washington, USA) using the immunoSEQ survey level assay.
Immunoseq survey level assay
Manufactured by Adaptive Biotechnologies
Sourced in United States
The ImmunoSEQ survey level assay is a DNA sequencing-based tool developed by Adaptive Biotechnologies. It is designed to provide a comprehensive overview of the adaptive immune repertoire, including the identification and quantification of T-cell and B-cell populations.
Automatically generated - may contain errors
5 protocols using immunoseq survey level assay
1
CSF Immunoglobulin Variable Sequencing
2
TCRβ Sequencing of Tumor and Spleen
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Tumors and spleens were isolated 14 days after tumor injection followed by genomic DNA extraction using the DNeasy Blood and Tissue Kit (QIAGEN) according to manufacturer’s protocol. TCRβ sequencing was performed using the ImmunoSEQ survey level assay (Adaptive Biotechnologies). Sequencing data were analyzed using the ImmunoSEQ analyzer (Adaptive Biotechnologies) and the Vegan package in R.
3
Analyzing Tumor Infiltrating T Cells
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On day 14 after tumor inoculation, B16F10 tumors and spleens were isolated. Genomic DNA was extracted using the DNeasy Blood and Tissue Kit (QIAGEN). TCRβ sequencing was performed using the ImmunoSEQ survey level assay (Adaptive Biotechnologies). Sequencing data was analyzed using the ImmunoSEQ analyzer (Adaptive Biotechnologies).
4
Isolation and Sequencing of CD8+ T cells
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Tumor genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen). CD8α+ R9F-specific T cells were purified by FACS using R9F-dextramer reagent, anti-CD8α and anti-CD3. The cells were pelleted, frozen at -80 °C and sent to Adaptive Biotechnologies. The TCRβ locus was sequenced using the ImmunoSEQ survey level assay by Adaptive Biotechnologies (Seattle, WA). TCRβ sequencing was analyzed using the ImmunoSEQ Analyzer (Adaptive Biotechnologies).
5
CSF and Blood Immune Profiling
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We collected between 20 and 25 ml CSF by lumbar puncture and approximately 60 ml blood by venipuncture at each time point. Mononuclear cells were isolated, and samples were prepared as previously described (Johansen, Vartdal, 2015) . Multiplex PCR of IGHV cDNA and high-throughput sequencing on the Illumina MiSeq system were performed by Adaptive Biotechnologies (USA) using the immunoSEQ survey level assay (coverage £ 100,000 B cells) (Larimore et al., 2012) . The survey level depth was chosen for both CSF and blood mononuclear cells in order to make valid comparisons across the two compartments. The sequencing raw data were preprocessed and normalized as previously described (Johansen, Vartdal, 2015) .
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