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| Status |
Public on Apr 18, 2021 |
| Title |
PBMC_CD4posIL17neg_TCRb |
| Sample type |
SRA |
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| Source name |
sorted IL-17-CD4+ T cells after M3R peptide stimulation for 9 days
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| Organism |
Homo sapiens |
| Characteristics |
cell type: lineage-negative (CD11b, CD19, NK1.1 and Ter119) and CD4+ CD8- T cells in PBL
strain: pSS patient
tissue: peripheral blood
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Salivary gland biopsy sample was taken at the time of diagnosis, and stored in RNA later solution at -30℃. Peripheral blood mononuclear cells were collected from M3R reactive Th17 positive pSS patient, and cultured with M3R peptide for 8 days. After stimulating with M3R peptide for 12hrs, IL-17 secreting CD4+ T cells were identified by Flow cytometry, using MACS cytokine secretion detection kit. Detected CD4+ IL-17+ T cells were purified by cell sorting.
TCR sequencing libraries for next generation sequencing were prepared from the mRNA of FACS-sorted IL-17 producing or non-producing CD4+ T cells. PolyA RNAs were isolated and amplified from sorted T cells according to a previous report (Shichino, et al, JCI Insight 2019). Human TCR beta chain cDNAs are amplified and fragmented according to a previous report (Shitara et al J Immunother Cancer 2019). Final TCR libraries, whose lengths were 200–300 base pairs, were pooled and sequenced using an Ion 540 Chef kit, an Ion 540 Chip kit, and an Ion Genestudio S5 Sequencer (Thermo Fisher Scientific) according to the manufacturer’s instructions, except the input library concentration (65 pM) and flow number (500).
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Ion Torrent S5 |
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| Data processing |
Library strategy: TCR-seq
Adapter trimming and quality filtering of sequencing data were performed by using PRINSEQ-0.20.4. Sequencing data were processed by MiXCR-3.0.5. Filtered reads were aligned to reference mouse TCR V/D/J sequences and assembled into TCR clonotypes using "analyze amplicon" command with the following parameters: -s hsa, --starting-material rna, --5-end no-v-primers, --3-end c-primers, --adapters no-adapters, --receptor-type trb, --region-of-interest CDR3, --only-productive, --align "-OvParameters.geneFeatureToAlign=VTranscript", --align "-OvjAlignmentOrder=JThenV", --assemble "-ObadQualityThreshold=10", --assemble "-OseparateByV=true", --assemble "-OseparateByJ=true".
List of final clones were converted into VDJtools format and analyzed by VDJtools-1.2.1.
Genome_build: ImMunoGeneTics (IMGT) reference sequences of human TRB
Supplementary_files_format_and_content: text files in VDJtools format include read count and frequency, complementarity determining region 3 (CDR3) nucleotide and amino acid sequences, Variable(V)/ Diversity(D)/ Joining(J) segment names, and V, D and J segment boundaries within CDR3 nucleotide sequence of individual clones
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| Submission date |
Apr 20, 2020 |
| Last update date |
Apr 19, 2021 |
| Contact name |
Shigeyuki Shichino |
| E-mail(s) |
s_shichino@rs.tus.ac.jp
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| Organization name |
Tokyo University of Science
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| Department |
Department of Molecular regulation of Inflammatory and Immune Diseases
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| Street address |
2641, Yamasaki, Noda-shi
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| City |
Chiba |
| ZIP/Postal code |
278-0022 |
| Country |
Japan |
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| Platform ID |
GPL23934 |
| Series (1) |
| GSE148966 |
Identification of M3 muscarinic acetylcholine receptor reactive Th17 cells in patients with primary Sjögren’s syndrome |
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| Relations |
| BioSample |
SAMN14646283 |
| SRA |
SRX8143633 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4486773_PMBC_CD4posIL17neg_TCRB.txt.gz |
1.4 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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