 |
 |
GEO help: Mouse over screen elements for information. |
|
Status |
Public on Mar 21, 2024 |
Title |
KO Th17 with NAC biol rep 3 |
Sample type |
SRA |
|
|
Source name |
CD4+T cells
|
Organism |
Mus musculus |
Characteristics |
cell type: CD4+T cells genotype: SPTLC1 defecient (KO) treatment: KO Th17 with NAC
|
Treatment protocol |
For Th17 differentiation, naïve CD4+ T cells at 1 million cells/ml concentration were activated by plate bound anti-CD3 (5 mg/ml) and soluble anti-CD28 (2 mg/ml) for 4 days along with hTGF-β1 (5 ng/ml), IL-6 (40 ng/ml), anti-IL-4 (10 mg/ml) and anti-IFN-γ (10 mg/ml).
|
Growth protocol |
Cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin and 50 mM 2-mercaptoethanol referred as complete medium (CM)
|
Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was extracted for each of the 3 bioological replicates from WT Th17, KO Th17, WT Th17 with NAC, KO Th17 with NAC, WT Th17 with 3-KDS, KO Th17 with 3-KDS, WT naive and KO naive CD4+ T cells using Trizol and RNA-Clean and concentrater column. About 1.3 ug of RNA in 25ul was submitted for bulk RNAseq using Illumina machine (HWI-ST1276). Total RNA sample quality was measured using Nanodrop (OD260/280), gel electrophoresis and Agilent 2100 to measure RNA integrity. Susequently, mRNA was enriched using oligo(dT) beads, fragmented, cDNA was synthesized using random hexamers, followed by the second strand cDNA synthesis using dTTP. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries was pooled and sequenced on Illumina platforms, according to effective library concentration and data amount.
|
|
|
Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NovaSeq 6000 |
|
|
Data processing |
Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality. Index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5 featureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene Assembly: Mus Musculus(GRCm38/mm10) Supplementary files format and content: gene_count.xls Supplementary files format and content: gene_fpkm.xls
|
|
|
Submission date |
Jan 30, 2024 |
Last update date |
Mar 21, 2024 |
Contact name |
Thiruvaimozhi Abimannan |
E-mail(s) |
thiruvaimozhi.abimannan@nih.gov
|
Phone |
3018465652
|
Organization name |
National Cancer Institute
|
Department |
CDBL
|
Lab |
Jairaj Acharya Lab (SPHINGOLIPID AND PHOSPHOLIPID SIGNALING SECTION)
|
Street address |
Building 560, Room 22-6
|
City |
Frederick |
State/province |
MD |
ZIP/Postal code |
21702 |
Country |
USA |
|
|
Platform ID |
GPL24247 |
Series (1) |
GSE254602 |
Effect of deletion of SPTLC1 on gene expression and differentiation of mouse Th17 cells in vitro |
|
Relations |
BioSample |
SAMN39674637 |
SRA |
SRX23452899 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
|
|
|
|
 |