|
| Status |
Public on Dec 22, 2021 |
| Title |
RNA-Seq Day3_1_S10 |
| Sample type |
SRA |
| |
|
| Source name |
P14 CD8+ T cells
|
| Organism |
Mus musculus |
| Characteristics |
sample group: CD8_In_Vitro_Timecourse
timepoint: Day 3
condition: in vitro WT, 3 days post antiCD3/28 stimulation
dox_added_to_fibroblast: NA
batf_expressed_in_fibroblast: NA
irf4_expressed_in_fibroblast: NA
tbet_expressed_in_fibroblast: NA
runx3_expressed_in_fibroblast: NA
|
| Treatment protocol |
In vitro P14 CD8+ T cells: on day 6, cells were left untreated or activated with 50 ng/ml PMA and 1 uM ionomycin for 3 hours before harvest for RNA-seq. In vivo P14 CD8+ T cells: no treatment. NIH/3T3 fibroblast: cells were transduced with doxycycline-inducible TF-expressing lentivirus. Fibroblasts were left untreated or treated with 4 ug/ml doxycline for 72 hours for TFs induction before harvest for RNA-seq.
|
| Growth protocol |
In vitro P14 CD8+ T cells: naïve CD8+ T cells were isolated with Miltenyi kit (Cat#130-096-543), stimulated with anti-CD3 (2 ug/ml) and anti-CD28 (2 ug/ml) and cultured in RPMI (supplemented with HEPES, NEAA, sodium pyruvate, 2-Mercaptoethanol, 10% FBS) in the presenece of 100 U/ml IL-2. In vivo P14 CD8+ T cells were sorted directly from LCMV infected mice. NIH/3T3 fibroblast: cells were cultured in DMEM supplemented with 10% FBS.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were lysed and RNA was extracted with Qiagen RNeasy Plus Kits (Qiagen Cat#74034) according to manufacturer's protocol.
mRNA was isolated with NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, E7490) and the RNA-seq libraries were generated with NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB, E7760) according to manufacturer’s instructions.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
| |
|
| Description |
Day3_1_S10
|
| Data processing |
All RNA-seq reads were trimmed using Trimmomatic (60) to remove primer and low-quality bases.
Reads were then passed to FastQC (61) to check the quality of the trimmed reads.
The single-end reads were then aligned to the Mus_musculus.GRCm38.82 transcriptome from Ensembl using RSEM (62) with the parameters “--num-threads 4 --bowtie2 --sampling-for-bam --output-genome-bam --sort-bam-by-coordinate --sort-bam-memory-per-thread 1G --estimate-rspd --fragment-length-mean 200 --fragment-length-sd 80’.
Normalized counts obtained using DESeq2, applied to each of the three datasets separately.
Genome_build: Mus_musculus.GRCm38.82 transcriptome
Supplementary_files_format_and_content: Tab-delimited files of normalized counts for each replicate
|
| |
|
| Submission date |
Dec 21, 2021 |
| Last update date |
Dec 22, 2021 |
| Contact name |
Jim Kaminski |
| E-mail(s) |
[email protected]
|
| Organization name |
Boston Children's Hospital
|
| Street address |
1 Blackfan Circle
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL21626 |
| Series (2) |
| GSE192389 |
Batf-mediated Epigenetic Control of Effector CD8+ T Cell Differentiation (RNA-Seq) |
| GSE192390 |
Batf-mediated Epigenetic Control of Effector CD8+ T Cell Differentiation |
|
| Relations |
| BioSample |
SAMN24281947 |
| SRA |
SRX13473982 |
