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Sample GSM4629065 Query DataSets for GSM4629065
Status Public on Oct 28, 2020
Title Single-cell RNA-Seq of Tumor-infiltrating T cells
Sample type SRA
Source name Tumor-infiltrating T cells in YTN2 and YTN16
Organism Mus musculus
Characteristics strain: C57BL/6
tumor type: gastric cancer
cell type: T cells
Extracted molecule polyA RNA
Extraction protocol The single-cell libraries were prepared using the Flow-cell devices composed of multiple vertical flow array chips) (VFACs, Shirai et al, Scientific Reports, 6, srep36014, 2016). VFAC (1 mm square) contains 100 microchambers packed with 1~2 x105 magnetic beads (1 µm in diameter) immobilized 7.5~15 x109 RT probes. RT probes consisted of 18nt of poly-T sequence, 6 nt of cell identifier (Cell-ID), 7nt of random sequence as UMIs(Unique Molecular Identifiers), and 30nt of common sequence (CS) for amplification (Supplementary Table 2). The procedures from cell captureing to cDNA synthesis were performed in the flow-cell device. In brief summary, FACS-sorted tumor-infiltrating lymphocytes isolated from YTN2 and YTN16 tumor tissues were washed once with PBS, and resuspended in PBS containing 90 copies/μL of spiked cRNA to a final concentration of 80 cells/μL. After adding 80 cells per a VFAC, the flow-cell device was connected to a vacuum pump (Ulback KOKI, Inc.) for appling negative pressure to the rear side of the VFACs, and single-cells were captured onto laser 4-micro-meter holes in diameter fabricataed at top of the microchambers respectively. Following cell lysis reagent, 4.5 μL of RT reagent (a mixture of 1.0 μL of 5 x FS buffer, 1.0 μL of 100 mM DTT, 1 μL of 10 mM dNTPs, 0.3 μL of 10 % Tween20, 0.4 μL of RNase OUT, and 0.4 μL of Super Script III (Thermo Fisher Scientific)) was added per a VFAC in the flow-cell device, and reverse transcription was performed for 50 minutes at 50 ℃ in a thermostatic incubator. Each VFAC was took out from the flow-cell device into a 0.2 mL tube containing 100 μL of resuspension buffer (50 mM Tris (pH 8.0) containing 0.1% Tween 20). Magnetic beads on which single-cell cDNA libraries were constructed were collected from microchambers of VFAC using a neodymium magnet, and washed twice with 50 μL of resuspend buffer. After exonuclease I treatment, 1st PCR was performed with primers listed in Supplementary Table 3, and the product was purified with Ampure XP (Beckman Coulter). Following 2nd PCR was performed with primers listed in Supplementary Table 4, the product was purified with Ampure XP. 3rd PCR was performed with primers containing illumina-tag sequences and index (chip-ID) listed in Supplementary Table 5, and the product was purified with Ampure XP. The single-cell libraries were analyzed with 2100 Bioanalyzer high sensitivity DNA kit (Agilent), to confirm a fragment distribution within a size range of 461–667 bp. More than 3M reads for each chip was assigned to get sufficient deep sequencing data. Libraries of 3 or 4 chips were pooled before sequencing. Infomations of cells on each chip were shown in Supplementary Table 6. The final concentration of each 3rd PCR product should be diluted with 10 mM Tris:HCl (pH7.5) with 0.1 % tween20 to be 3.2 nM. According to the user manual of Miseq, denature is carried out. Phi X control is mixed at 10%. Paired-end sequencing (read 1 = 60bp, read 2 = 90bp) was carried out using Miseq (150 cycle (v3) reagents).
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina MiSeq
Data processing CEL-Seq2 was used for UMI counting. UMI count data for each micro-chamber was normalized with UMIs for spked cRNA of luciferase.
Supplementary_files_format_and_content: Normalized UMI counts
Submission date Jun 20, 2020
Last update date Oct 28, 2020
Contact name Koji Nagaoka
Organization name The Tokyo University Hospital
Department Department of Immunotherapeutics
Street address 7-3-1 Hongo, Bunkyo-ku
City Tokyo
ZIP/Postal code 113-8655
Country Japan
Platform ID GPL16417
Series (1)
GSE152888 Targeted single cell RNA sequencing of T cells infiltrated in mouse gastric cancer.
BioSample SAMN15332179
SRA SRX8588604

Supplementary file Size Download File type/resource
GSM4629065_Processed_data_normalized.txt.gz 97.4 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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