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Sample GSM5462684

Query DataSets for GSM5462684

Status

Public on Jul 22, 2021

Title

Control-4

Sample type

SRA

Source name

CD8+ T cells

Organism

Mus musculus

Characteristics

strain: C57BL/6
gender: Female
age: Six- to eight-week-old
tissue: Inguinal lymph nodes and spleen

Treatment protocol

Four mice were used in the analysis. On day 25, the lymph nodes and spleen were gently disrupted and filtered through a 40 μm nylon cell strainer to make single-cell suspensions. Cells were treated with RBC lysis buffer before use. For each mice, Thy1.1-negative endogenous CD8+ T cells were isolated from the lymphoid cells with a CD8 isolation kit and Thy1.1 microbeads. Then, the IL-18Rαhi-enriched cells were positively selected with PE-labeled IL-18Rα antibody and PE microbeads. The cells that passed through the column were collected and used as a control for comparison with IL-18Rαhi-enriched cells.

Growth protocol

On day 0, six- to eight-week-old C57BL/6 female mice were intradermally inoculated with B16F10 (2E+05). On day 3, pre-conditioning lymphodepletion was induced via intraperitoneal injection of 300 mg/kg cyclophosphamide. On day 5, Pmel-1 cells (2E+06) were isolated from the splenocytes of Thy1.1 Pmel-1 transgenic mice (Thy1a/Cy Tg(TcraTcrb)8Rest/J; expressing transgenic TCR specific for gp10025–33 and congenic Thy1.1 antigen) and injected into tumor-bearing mice. Starting on day 5, recombinant human IL-2 (10,000 IU) was intraperitoneally injected daily for 3 days. Starting from day 10, anti-CD4 treatment group involved weekly intraperitoneal injections of 200 μg anti-mouse CD4 monoclonal antibody (clone: GK1.5) for 3 weeks.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted from the isolated cells using Trizol reagent.
cDNA libraries were prepared for 150 bp paired-end sequencing using TruSeq Stranded mRNA sample preparation kit. In brief, the mRNA molecules were purified and fragmented from 1 μg total RNA using oligo (dT) magnetic beads. The fragmented mRNAs were synthesized as single-stranded cDNAs using random hexamer primers. Double-stranded cDNA was prepared using this as a template for second strand synthesis. After sequential end repair, A-tailing, and adapter ligation, the cDNA libraries were amplified via PCR. The quality of these cDNA libraries was evaluated using the Agilent 2100 BioAnalyzer and they were quantified using the KAPA library quantification kit according to the manufacturer’s protocol.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

IL18Rαhi-depleted flowthrough-4

Data processing

Low quality reads were filtered according to the following criteria: reads containing more than 10% of skipped bases (marked as ‘N’s), reads containing more than 40% of bases whose quality scores were less than 20, and reads with average quality score less than 20.
The filtered reads were mapped to the reference genome related to Mus musculus using the aligner TopHat.
Gene expression level was determined using Cufflinks v2.1.1 of the gene annotation database of the species. To improve the accuracy of the measurement, multi-read-correction and frag-bias-correct options were applied. All other options were set to default values.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files that includes FPKM values of all samples

Submission date

Jul 20, 2021

Last update date

Jul 22, 2021

Contact name

Chungyong Han

Organization name

National Cancer Center

Street address

323 Ilsan-ro, Ilsandong-gu

City

Goyang-si