|
| Status |
Public on Sep 22, 2023 |
| Title |
mPiC_d6_1 |
| Sample type |
SRA |
| |
|
| Source name |
n/a
|
| Organism |
Mus musculus |
| Characteristics |
cell line: n/a
cell type: CD8 T cells
genotype: Slc25a3-fl/fl Cd4-cre
time: Day 6
|
| Treatment protocol |
Day0 samples were directly purified from spleen and lymph node. Day2 samples were purified from naïve mice and stimulated with anti-CD3/CD28. Day6 samples were acitivated as day2 samples followed with a 4 days treatment of IL-2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
1x10^6 T cells were harvested in RNAprotect Cell Reagent and stored at -80°C o/n before total RNA was extracted using the RNeasy Plus Micro Kit (both Quiagen)
RNA quality and quantity were checked using a 2100 Bioanalyzer with the RNA 6000 Nano kit (Agilent Technologies) and RNA Pico kit depending on the sample concentration. The RIN for all samples was >7. cDNA libraries suitable for sequencing were prepared from 5 ng of total RNA with SMART-Seq v4 Ultra Low Input RNA Kit (Takara) according to manufacturer’s instructions (1/4 volume). Libraries were quantified by QubitTM 3.0 Fluometer (ThermoFisher) and quality was checked using 2100 Bioanalyzer with High Sensitivity DNA kit (Agilent). 0.5 ng of each library was subjected to a tagmentation-based protocol (Nextera XT, Illumina) using a quarter of the recommended reagent volumes. Libraries were quantified again by QubitTM 3.0 Fluometer (Thermo Fisher) and quality was checked using 2100 Bioanalyzer with High Sensitivity DNA kit (Agilent) before pooling. Sequencing of pooled libraries, spiked with 1% PhiX control library, was performed at 19-36 million reads/sample in single-end mode with 75 nt read length on the NextSeq 500 platform (Illumina) with 1 High Output Kit v2.5.
Sequencing of pooled libraries, spiked with 1% PhiX control library, was performed at ~20 million reads/sample in single-end mode with 75 nt read length on the NextSeq 500 platform (Illumina) with 1 High Output Kit v2.5. Demultiplexed FASTQ files were generated with bcl2fastq2 v2.20.0.422 (Illumina).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
DESeq2_comparison_mPiC_d2_vs_WT_d2_table
|
| Data processing |
Demultiplexed FASTQ files were generated with bcl2fastq2 v2.20.0.422 (Illumina). To assure high sequence quality, Illumina reads were quality- and adapter-trimmed via Cutadapt [1] version 2.5 using a cutoff Phred score of 20 in NextSeq mode, and reads without any remaining bases were discarded (command line parameters: --nextseq-trim=20 -m 1 -a CTGTCTCTTATACACATCT). Processed reads were subsequently mapped to the mouse genome (GRCm38.p6 primary assembly and mitochondrion) using STAR v2.7.2b with default parameters based on RefSeq annotation version 108.20200622 for GRCm38.p6
Read counts on exon level summarized for each gene were generated using featureCounts v1.6.4 from the Subread package. Multi-mapping and multi-overlapping reads were counted non-strand-specific with a fractional count for each alignment and overlapping feature (command line parameters: -s 0 -t exon -M -O --fraction). The count output was utilized to identify differentially expressed genes using DESeq2 version 1.24.0. Read counts were normalized by DESeq2 and fold-change shrinkage was applied by setting the parameter “betaPrior=TRUE”. Differences in gene expression were considered significant if padj < 0.05.
Assembly: GRCm38.p6 primary assembly and mitochondrion
Supplementary files format and content: DESeq2_comparison
|
| |
|
| Submission date |
Aug 30, 2022 |
| Last update date |
Sep 22, 2023 |
| Contact name |
hao wu |
| E-mail(s) |
dududuoq@gmail.com
|
| Phone |
015903894761
|
| Organization name |
institute for systemsimmunology, wurzburg university
|
| Lab |
AG Martin Väth
|
| Street address |
Versbacher stress 7-9
|
| City |
Würzburg |
| ZIP/Postal code |
97078 |
| Country |
Germany |
| |
|
| Platform ID |
GPL19057 |
| Series (1) |
| GSE212298 |
RNA-seq of wild type and Slc25a3-deficient CD8 T cells |
|
| Relations |
| BioSample |
SAMN30592579 |
| SRA |
SRX17321027 |
