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| Status |
Public on Feb 24, 2020 |
| Title |
JHU083 CD8TIL5 |
| Sample type |
SRA |
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| Source name |
sorted CD11b-CD11c-B220-CD45+TCRbeta+CD44+CD8+ TIL
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell type: sorted CD11b-CD11c-B220-CD45+TCRbeta+CD44+CD8+ TIL
tumor model: 5X10^5 cells MC38 (murine colon carcinoma) s.c. into right flank of C57BL/6 mice
treatment: JHU083, pro-drug of 6-Diazo-5-oxo-L-norleucine (glutamine antagonist) on day 14-18 post-implantation (0.3mg/kg/day) then harvested and sorted on day 18
grouping: B
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| Treatment protocol |
Sorting with FACSAria Fusion
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| Growth protocol |
5X10^5 cells MC38 (murine colon carcinoma) s.c. into right flank of C57BL/6 mice
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Sorted into TRIZOL (LS); RNeasy Mini Kit (QIAGEN)
Trizol samples were sent to Admera Health for RNA extraction (RNeasy Mini Kit (QIAGEN)) and to process library construction and data analysis. Paramagnetic beads coupled with oligo d(T) are combined with total RNA to isolate poly(A)+ transcripts based on NEBNext® Poly(A) mRNA Magnetic Isolation Module manual. Prior to first strand synthesis, samples are randomly primed (5´ d(N6) 3´ [N=A,C,G,T]) and fragmented based on manufacturer’s recommendation (NEBNext® Ultra™ RNA Library Prep Kit for Illumina®). The first strand is synthesized with the Protoscript II Reverse Transcriptase with a longer extension period (40 minutes for 42◦C). All remaining steps for library construction were used according to the NEBNext® Ultra™ RNA Library Prep Kit for Illumina®. Illumina 8-nt dual-indices were used.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
DRUG5
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| Data processing |
Sequencing was performed on NextSeq 500/550 V2 Systems 75 cycles (1x75bp). NextSeq 500/550 V2 Systems suite version 2.1.2, bcl2fastq2 version 2.17.1.14. Data processing Illumina NextSeq 500/550 V2 reads were trimmed and clipped for quality control based on sliding windows considering quality and length thresholds to determine the quality at which it is sufficiently low to trim the 3′-end of reads and also determines the quality at which it is sufficiently high enough to trim the 5′-end of reads. This approach finds and removes adapter sequences, primers, poly-A tails and other types of unwanted sequence from high-throughput sequencing reads. Quality Threshold < 30
The pre-processing operations included a quality check (FastQC), removal of adapter content (used during sequencing), quality thresholding (remove any bad quality reads)
Read quality was checked for each sample using FastQC 0.11.2.
High-quality reads were then imported into a fast splice junction mapper for RNA-Seq reads and this aligns RNA-Seq reads to genomes using the ultra high-throughput short read aligner. The mapping results were then analyzed to identify splice junctions between exons, for alignment into BAM files.
BAM files were imported into the RNA-Seq pipeline by the Transcriptomic Analysis Pipeline (proprietary pipeline designed in-house).
Reads were mapped to the reference genome. This aligns RNA-Seq reads to the genome using a short read aligner and discovers the splice sites.The resulting alignment files were used to generate a transcriptome assembly. TopHat (v 2.1.1) used
The major stages of analysis included read mapping to the latest reference genome (GRCM38), assembly of the mapped reads to identify expressed transcripts and genes, followed by quantification and comparison of the assembled transcriptomes between cells.
Gene and transcript expression between the comparing RNASeq samples were quantified and reported as FPKM (Fragments Per Kilobase of transcript per Million mapped reads) units
Genome_build: GRCM38
Supplementary_files_format_and_content: Excel file with FPKM
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| Submission date |
Sep 23, 2018 |
| Last update date |
Feb 24, 2020 |
| Contact name |
Jonathan D. Powell |
| E-mail(s) |
poweljo@jhmi.edu
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| Organization name |
Johns Hopkins University
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| Department |
Oncology
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| Street address |
1650 Orleans Street
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| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21231 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (1) |
| GSE120345 |
RNA-sequencing on sorted infiltrating CD8 T cells from vehicle or JHU083, pro-drug of 6-Diazo-5-oxo-L-norleucine (glutamine antagonist) treated MC38 (colon cancer) tumor bearing mice |
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| Relations |
| BioSample |
SAMN10106040 |
| SRA |
SRX4730164 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3398550_JHU5.xlsx |
11.1 Mb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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