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| Status |
Public on Apr 26, 2022 |
| Title |
Day 4 exhausted OT-I-1 |
| Sample type |
SRA |
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| Source name |
T cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: T cells
genotype: OT-I
treatment: N/A
time: Day 4
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| Treatment protocol |
N/A
N/A
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| Growth protocol |
C57Bl6/J mice bearing B78ChOVA melanomas received adoptively transferred OT-I T cells 4 or 14 days prior to sacrifice. Tumor-infiltrating CD44+ OT-I T cells were isolated by FACS sorting in lysis buffer for RNAseq. Naïve OT-I T cells were directly isolated from lymph nodes of tumor-naive OT-I mice. Effector OT-I T cells were generated in vitro upon 5 days of antigenic stimulation.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were directly sorted in lysis buffer, RNA was extracted using Dynabead Direct.
mRNA was converted into amplified cDNA using the Tecan Ovation RNA-Seq System V2 kit, following the manufacturer guidelines. The dsDNA is tagmented, amplified and undergoes clean up with AMPure XP bead, using the Illumina Nextera XT DNA Library Prep Kit. The resulting sequencing library is QC’d using an Agilent Bioanalyzer HS DNA chip to assess fragment size distribution and concentration. Libraries were pooled prior to single-end sequencing on and Illumina MiSeq/MiniSeq to ensure quantify library complexity. Libraries with less than 10 percent of the reads aligned to coding regions, or fewer than 1,000 unique reads in total were rejected. The validated libraries were re-pooled based on the percentage of reads in coding regions and submitted for sequencing.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Raw fastq reads were QC’d and trimmed to remove adapter contamination, and poly-G artifacts using using fastp version 0.19.6. Reads with fewer than 20bp post-trimming were discarded.
Trimmed reads were aligned to the GRCm38 reference sequence annotated with Gencode V25 using STAR version 2.6.1b (Dobin et al., 2013) with the following parameters (--quantMode GeneCounts –outFilterMismatchNoverLmax 0.04 --alignIntronMax 100000 --alignMatesGapMax 100000 --alignSJDBoverhangMin 10 --alignSJstitchMismatchNmax 5 -1 5 5 --chimSegmentMin 12 --chimJunctionOverhangMin 12 --chimSegmentReadGapMax 3 --chimMultimapScoreRange 10 --chimMultimapNmax 10 --chimNonchimScoreDropMin 10 --peOverlapNbasesMin 12 --peOverlapMMp 0.1)
Assembly: GRCm38
Supplementary files format and content: tab-delimited text files with CPM values for each Sample as reported by the STAR aligner.
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| Submission date |
Apr 19, 2022 |
| Last update date |
Apr 27, 2022 |
| Contact name |
Arjun Arkal Rao |
| E-mail(s) |
arjunarkal.rao@ucsf.edu
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| Organization name |
University of California, San Francisco
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| Department |
CoLabs
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| Lab |
Data Sciences CoLab
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| Street address |
505 Parnassus Ave, S-447
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| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94143 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE201071 |
Spatiotemporal co-dependency between macrophages and exhausted CD8+ T cells (RNA-Seq) |
| GSE201074 |
Spatiotemporal co-dependency between macrophages and exhausted CD8+ T cells |
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| Relations |
| BioSample |
SAMN27642752 |
| SRA |
SRX14920048 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6050182_d4_iso_1.ReadsPerGene.out.tab.gz |
332.8 Kb |
(ftp)(http) |
TAB |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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