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SRX4125977: GSM3155143: N01_unstim_all_beta; Homo sapiens; OTHER
1 ILLUMINA (Illumina HiSeq 1500) run: 2.3M spots, 798.9M bases, 429.8Mb downloads

Submitted by: NCBI (GEO)
Study: Antigen-specific T-cell receptor signatures of cytomegalovirus infection
show Abstracthide Abstract
We immunosequenced peptide-stimulated PBMCs from 8 CMV-positive donors to identify virus-specific T cell receptors. We also sequenced ex vivo T cell repertoires of multiple CMV-postive and CMV-negative donors to compare the availability of CMV-specific TCRs in virus carriers and uninfected subjects. Overall design: Blood samples from healthy anonymous donors were obtained from the "Institut fu¨r Transfusionsmedizin Ulm ", Germany. PBMCs were isolated by standard Ficoll gradient centrifugation and mRNA was isolated either directly or after 10-day stimulation with an immunogenic CMV peptide. For validation of our short-term stimulated TCRs, PBMCs from 3 different donors were stimulated with autologous transformed B cells (mini-LCLs) for 23 or 30 days. These mini-LCLs expressed one of the CMV antigens pp65 or IE1 together with EBV antigens, or EBV antigens alone (control mini-LCLs). TCR-enriched sequencing libraries were obtained using TCR beta chain-specific primers with Illumina adapter sequence overhangs. After Sequencing on an Illumina HiSeq1500 device, raw data was demultiplexed, quality filtered, and TCR repertoires were generated by alignment and grouping of individual reads.
Sample: N01_unstim_all_beta
SAMN09261081 • SRS3340440 • All experiments • All runs
Organism: Homo sapiens
Library:
Instrument: Illumina HiSeq 1500
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: PAIRED
Construction protocol: RNA extraction was performed on bulk cells (either unsorted or CD8-enriched using MACS) with the Qiagen RNeasy kit. 1 μg RNA per sample was reversely transcribed to cDNA with the QuantiTect Reverse Transcription Kit (Qiagen) using a primer designed to target both the Cβ1 and Cβ2 regions of the TCR RNA. cDNA was amplified in two subsequent PCRs using Pfu DNA polymerase (Thermo Scientific). The first PCR was a multiplex PCR comprising 42 distinct forward primers that bind to the Vβ region and cover all possible human TCR Vβ segments, and a reverse primer that anneals to the Cβ1 and Cβ2 regions. Forward and reverse primers carried sequences complementary to Illumina Read 2 and Read 1 primer sequences. In the second PCR, index primers (NEBNext Multiplex Oligos for Illumina; New England BioLabs) were used to attach barcodes and the i5 and i7 adapters for binding to the Illumina flow cell (Supplementary Fig. 2). After each PCR step, the PCR product was purified using Agencourt AMPure XP magnetic beads (Beckman Coulter). The correct length and amount of the PCR products for sequencing was confirmed using the Agilent DNA 1000 Kit and the Bioanalyzer 2100 (Agilent). To enhance the nucleotide diversity of TCR reads facilitating cluster recognition and avoiding artifacts in q-score calibration and base calling, three different forms of the reverse primer, with 0, 1 or 2 arbitrary nucleotides between the Cβ-binding sequence and the Illumina Read 1 sequence were used. All primers were used in equimolar amounts (a total of 10μM; for primer sequences see Supplementary Table 1). The first PCR consisted of only 10 amplification cycles to minimize PCR amplification bias.
Experiment attributes:
GEO Accession: GSM3155143
Links:
Runs: 1 run, 2.3M spots, 798.9M bases, 429.8Mb
Run# of Spots# of BasesSizePublished
SRR72194292,314,373798.9M429.8Mb2018-11-23

ID:
5611538

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