The largest database of trusted experimental protocols

29 protocols using ImmunoSEQ Analyzer 3.0

Analysis of the sequences was performed on the immunoSEQ ANALYZER 3.0 (Adaptive biotechnologies). T cell rearrangements that are differentially abundant between samples were detected using the Differential Abundance tool by two-sided binomial tests with Benjamini and Hochberg multiple test correction, q value < 0.01 was considered statistically significant.
+ Open protocol
+ Expand
Control and PCSK9KO B16F10 cells were inoculated as described above and on day 10 after inoculation they were collected for genomic DNA extraction. Genomic DNA was extracted using DNeasy Blood & Tissue kit (Qiagen) and submitted to Adaptive Biotechnologies for mouse TCRB CDR3 survey sequencing. About 2.6 μg of initial DNA was used as input for PCR reaction. Data were analyzed using Adaptive Biotechnologies online analysis platform ImmunoSEQ Analyzer 3.0.
+ Open protocol
+ Expand
Genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, Germantown, MD) per manufacturer’s instructions. The immunosequencing of the CDR3 regions of human TCRβ chains was performed using the ImmunoSEQTM Assay (Adaptive Biotechnologies, Seattle, WA)49 (link). Briefly, genomic DNA was amplified in an unbiased multiplex PCR assay with a mixture of primers (Adaptive Biotechnologies, Seattle, WA)49 (link) targeting the rearranged variable and joining segments and a high-throughput DNA sequencing was performed. The sequences were quantitated and analyzed to identify frequency of productive TCRβ CDR3 rearrangements in samples using the ImmunoSEQ Analyzer 3.0 software provided by Adaptive Biotechnologies. Sample clonality and Morisita index were calculated using the ImmunoSEQ Analyzer 3.0 software. For other graphical presentations, data were exported as a tab-separated values file and graphs were plotted on Microsoft Excel. All associated Venn plots were created from the exported data using the webtool found at http://bioinformatics.psb.ugent.be/webtools/Venn/.
+ Open protocol
+ Expand
Effect of long-term treatment on B cell receptor repertoire was tested by sequencing BCR IgH chains. B-cell receptors were sequenced at high-throughput using bias-controlled multiplex PCR amplification from DNA isolated from PBMC of CHB patients (paired samples before and after treatment) by Adaptive biotechnologies (Seattle, USA). Data was analyzed using immunoSeq Analyzer 3.0 (Adaptive biotechnologies).
+ Open protocol
+ Expand
Control and PCSK9KO B16F10 cells were inoculated as described above and on day 10 after inoculation they were collected for genomic DNA extraction. Genomic DNA was extracted using DNeasy Blood & Tissue kit (Qiagen) and submitted to Adaptive Biotechnologies for mouse TCRB CDR3 survey sequencing. About 2.6 μg of initial DNA was used as input for PCR reaction. Data were analyzed using Adaptive Biotechnologies online analysis platform ImmunoSEQ Analyzer 3.0.
+ Open protocol
+ Expand

TCRB survey or deep sequencing was performed from gDNA by Adaptive Biotechnologies. Frozen, pelleted PBMCs or TILs (5 × 104 to 1 × 106 cells) were submitted for sequencing (see Table 2). Analysis of productive TCR rearrangements was performed using ImmunoSEQ Analyzer 3.0 (Adaptive Biotechnologies).
+ Open protocol
+ Expand
Genomic DNA was isolated using the QIAGEN DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA) either from PBMC or FACS sorted CLL-like (CD5+B220+) cells from the spleen of leukemic mice and 0.5–1 μg of genomic DNA per sample was used for sequencing. Sample data was generated using the immunoSEQ Assay (Adaptive Biotechnologies, Seattle, WA). The somatically rearranged mouse IGH CDR3 was amplified from genomic DNA using a two-step, amplification bias-controlled multiplex PCR approach (Carlson et al., 2013 (link); Robins et al., 2009 (link)). The first PCR consists of forward and reverse amplification primers specific for every V and J gene segment and amplifies the hypervariable complementarity-determining region 3 (CDR3) of the immune receptor locus. The second PCR adds a proprietary barcode sequence and Illumina adapter sequences. BCR repertoire analyses were performed using the immunoSEQ Analyzer 3.0 (Adaptive Biotechnologies). Sequences were subjected to analysis using IMGT/Vquest software to define all V, D and J genes as well as CDR3 sequences.
+ Open protocol
+ Expand
SPB cells were MACS-enriched and FACS-purified from the bone marrow of female 8-week old naïve adult WT, Il33−/−, and St2−/− mice. Genomic DNA (gDNA) was isolated using the QIAGEN DNeasy Blood & Tissue kit (Cat. #69504) and 0.5 μg of gDNA per sample was used for sequencing. Sequencing of the BCR hypervariable complementary determining region 3 (CDR3) was performed by the multiplex PCR-based immunoSEQ Assay (Adaptive Biotechnologies). BCR repertoire analyses were performed using the immunoSEQ Analyzer 3.0 (Adaptive Biotechnologies).
+ Open protocol
+ Expand
DNA was extracted from fresh frozen liver metastases and cryopreserved PBMCs before and after therapy with NHS-IL12 using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Venlo, Netherlands). TCR Vβ CDR3 sequencing (TCRseq) was performed at the NCI genomic core facility (Frederick, MD) using the survey (tumor) or deep (PBMCs) resolution Immunoseq platform (Adaptive Biotechnologies, Seattle, WA); analysis was performed using the ImmunoSeq ANALYZER 3.0 (Adaptive Biotechnologies). Repertoire size, a measure of TCR diversity, was determined by calculating the number of individual clonotypes represented in the top 25th percentile by ranked molecule count after sorting by abundance. Tumor-infiltrating lymphocyte (TIL) density was calculated from TCRseq data based on a human genome weight of 6.6 pg/cell: TIL density = (# productive templates) / (DNA input (pg) / 6.6).
+ Open protocol
+ Expand
CD3+ T cells were FACS-isolated from control PBMC T cells or iPSC-derived T cells, followed by gDNA extraction using the Qiagen DNeasy Blood & Tissue Kit (Qiagen, 69504). DNA samples were then subjected TCRB sequencing via immunoSEQ assay. (Adaptive Biotechnologies). TCR rearrangements, Vβgene usage, and CDR3 length were analyzed using the immunoSEQ Analyzer 3.0 software (Adaptive Biotechnologies).
+ Open protocol
+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!