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| Status |
Public on Jul 13, 2021 |
| Title |
RNA_P3_Tumor_1_0001 |
| Sample type |
SRA |
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| Source name |
tumor biopsy
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| Organism |
Homo sapiens |
| Characteristics |
age: 71
Sex: male
cancer type: esophageal
tissue: tumor biopsy
date: 44
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| Treatment protocol |
Patients with advanced gastrointestinal cancers with high TMB confirmed by Guardant360 (Guardant Health, Inc. Redwood City, CA), a 74-gene sequencing circulating tumor DNA assay, received intravenous nivolumab monotherapy of 360 mg every 3 weeks.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Tumor biopsy was conducted on before (day0) and after the treatment (day39-day55). Presence or absence of tumor tissue in biopsies was determined pathologically. Tumor biopsies were homogenized in TRIzol (Ambion, Carlsbad, CA, USA). RNA was extracted from each sample using the RNeasy mini kit (QIAGEN, Hilden, Germany, # 74106) and amounts and purity measured with the Agilent 2200 TapeStation (Agilent Technologies, Santa Clara, CA, USA). Five-micrograms of total RNA was diluted with 1 mL of cell lysis buffer and used for RNA sequencing.
PolyA RNAs were isolated according to a previous report with some modifications (GSE110711). Beads with containing cDNA was resuspended with whole transcript amplification mixture comprised 2 μL of 10 μM primer mix (trP1 and 3’WTA), 10.5 μL of dH2O, and 12.5 μL of KAPA Hifi Hotstart ReadyMix (KAPA Biosystems). The thermal cycling conditions were programmed as follows: denaturation at 95°C for 3 min, 12 cycles of denaturation for 20 sec at 98°C, annealing for 15 sec at 65°C and extension for 5 min at 72°C, followed by a final extension at 72°C for 5 min. Next, 25 μL of the first-PCR products were used for purification with an Agencort AM Pure XP kit (Bexkman-Coulter) at a 0.7:1 ratio of beads to sample, and eluted with 15 μL of DW. Fragmentasion and adaptor ligation were performed using 10-60ng of second-PCR product as a template with NEBNext FS DNA Library Prep Kit (New England Biolabs) with some modifications; NEBNext Adaptor for Illumina in “Adaptor Ligation” step was substituted by 1.5uM CS1 adaptor, and the reaction volume was 1/4 of the recommended in all steps. Adaptor-ligated DNA was added with 7.125 uL of 10x TE, then purified using Agencort AM Pure XP kit (Bexkman-Coulter) at a 0.4:1 ratio of beads to sample to remove large fragments, a 0.7:1 ratio of beads to sample to remove smaller fragments, and eluted with 20 μL of Tris-HCl (pH 8.0). The second PCR was carried out using barcoded primers to enrich the TCR cDNA flanked with sequencing adapters. The second PCR mixture consisted of 2.5 μL of 10 μM trP1 primer, 2.5 μL of 10 μM IonA-BC primer, 7.5 μL of template and 12.5 μL of NEBNext Ultra II Q5 Master Mix (accessory of NEBNext FS DNA Library Prep Kit). The thermal cycling conditions were programmed as follows: denaturation at 98°C for 30 sec, 11 cycles of denaturation for 10 sec at 98°C, annealing and extension for 1min 15 sec at 65°C, followed by a final extension at 65°C for 5 min. The PCR product was purified and subjected to size selection using Agencort AM Pure XP kit (Bexkman-Coulter) at a 0.7:1 ratio of beads to sample, and eluted with 20 μL of Tris-HCl (pH 8.0). Amplified TCR libraries were quantified using a KAPA SYBR Fast qPCR Kit (KAPA Biosystems, #KK4621) and size distribution was analyzed by agarose electrophoresis and SYBR Gold staining (Thermo Fisher Scientific, #S11494). Final RNAseq libraries, whose lengths were 200–300 base pairs, were pooled and sequenced using an Ion 540 Kit Chef, an Ion 540 Chip kit, and an Ion Genestudio S5 Sequencer (Thermo Fisher Scientific, # A27759, #A27766, # A38194) according to the manufacturer’s instructions, except the input library concentration (65 pM) and flow number (550).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Ion Torrent S5 |
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| Data processing |
Adapter trimming and quality filtering of sequencing data were performed using cutadapt v1.18. Filtered reads were mapped to hg38 Refseq RNA using Bowtie2-3.4.2 with the following parameters: -t -N 1 -D 200 -R 20 -L 20 -i S,1,0.50 --nofw. Tag numbers of each gene were quantified as the expression level of each gene.
Between-sample normalization was performed against raw count data using Microsoft R open 3.6.1 (https://mran.microsoft.com/open/) and TCC package (DDD-D method). Genes with fold change ≥4 and maximum expression ≥20 were identified as differentially expressed genes.
Co-expressed gene modules among differentially expressed genes were detected using the WGCNA package. For WGCNA, the power value was 19, the merge threshold value was 0.1, the threshold value for the output of co-expression interactions was 0.25, and other calculation settings were set to defaults. The genes in PR associated gene modules detected using WGCNA were further clustered into upregulated and downregulated genes in PR patients using hclust function of stats package.
Among genes upregulated in PR (1,606 genes), genes included in the term “T cell activation” in GO biological processes (GO) was extracted using Cytoscape 3.7.2 with ClueGO plugin (v2.5.5). We used versions of the GO term database and KEGG pathway term database that were current on Feb 27, 2019. If multiple biopsies are obtained at the same time point in patient, RNAseq and subsequent data processing were performed separately.
Genome_build: GRCh38
Supplementary_files_format_and_content: tab-delimited text files include raw tag-count values for each sample
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| Submission date |
Jul 16, 2020 |
| Last update date |
Jul 13, 2021 |
| Contact name |
Hiroyasu Aoki |
| E-mail(s) |
haoki-tky@rs.tus.ac.jp
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| Phone |
08013746493
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| Organization name |
Tokyo University of Science
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| Department |
Research Institute for Biomedical Sciences
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| Lab |
Division of Molecular Regulation of Inflammatory and Immune Diseases
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| Street address |
2669 Yamazaki
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| City |
Noda |
| State/province |
Chiba |
| ZIP/Postal code |
278-0022 |
| Country |
Japan |
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| Platform ID |
GPL23934 |
| Series (2) |
| GSE154538 |
Blood-Tumor overlapping TCR repertoire predicts the clinical responses of PD-1 blockade in patients with gastrointestinal cancer[RNA-Seq] |
| GSE154539 |
Blood-Tumor overlapping TCR repertoire predicts the clinical responses of PD-1 blockade in patients with gastrointestinal cancer |
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| Relations |
| BioSample |
SAMN15548479 |
| SRA |
SRX8742761 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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