|
| Status |
Public on Feb 29, 2024 |
| Title |
CD19_AAVS1_Donor_ND500 |
| Sample type |
SRA |
| |
|
| Source name |
Primary human T cells
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Primary human T cells
genotype: FOXO1 WT
|
| Treatment protocol |
Cells were treated the same, the only difference being which sgRNA was used for CRISPR knockout (sgAAVS1 vs. sgFOXO1)
|
| Growth protocol |
Primary human T cells were grown in AIM-V media supplemented with 5% FBS, 10mM HEPES, 1x Penicillin-Streptomycin-Glutamate, and 100U/mL recombinant human IL-2. T cells were activated with anti-CD3/CD28 beads immediately after thawing from liquid nitrogen. 48hrs post-activation, CRISPR-Cas9-based knockout was performed on the cells using a Lonza electroporation kit and electroporator. Then, at 48 and 72 hours post-activation were transduced with retroviruses containing CARs and transcription factors of interest. 96 hours post-activation, beads were removed; subsequently, T cells were resuspended at a concentration of 0.5e6 cells/mL every 2-3 days in the above described media. Since FOXO1 KO cells uniformly exhibited low CD62L surface expression, we used CD62L as a surrogate marker for FOXO1 editing and applied magnetic bead negative selection to enrich for CD62Llo/FOXO1KO cells using a StemCell EasySep magnetic sorting kit. 5e5-1e6 TCs were pelleted and flash frozen prior to RNA seq prep.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Pellets were thawed on ice and processed using either a RNEasy Plus Mini Kit or an AllPrep DNA/RNA Micro Kit (for simultaneous DNA and RNA isolation) (Qiagen) according to the manufacturer’s instructions. Total RNA was quantified using either a Qubit Fluorometer or a DeNovix DS-11 FX Spectrophotometer/Fluorometer
Sequencing was outsourced to Novogene - US. Libraries were prepared by the vendor and sequenced on a NovoSeq 6000 instrument.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
FOXO1 WT (AAVS1 KO) CD19.BBz CAR T cells were isolated on day 15 post activation. 5e5-1e6 TCs were pelleted and flash frozen prior to RNA seq prep.
|
| Data processing |
Fastq files were aligned to a mm10 transcriptome index with STAR
The abundance of transcripts was quantified using Salmon
Transcript-level abundance estimates were imported and summarized using tximport v1.16.1
Differential expression was determined using the DESeq2 package v1.28.11 in Bioconductor v.3.11.
Assembly: hg38
Supplementary files format and content: Raw and normalized counts(.csv).
|
| |
|
| Submission date |
Feb 08, 2024 |
| Last update date |
Feb 29, 2024 |
| Contact name |
Katherine Paige Mueller |
| E-mail(s) |
[email protected]
|
| Phone |
3037758187
|
| Organization name |
Children's Hospital of Philadelphia
|
| Department |
Pediatrics
|
| Lab |
Weber Lab
|
| Street address |
3501 Civic Center Blvd
|
| City |
Philadelphia |
| State/province |
Pennsylvania |
| ZIP/Postal code |
19104-3820 |
| Country |
USA |
| |
|
| Platform ID |
GPL24676 |
| Series (2) |
| GSE255410 |
FOXO1 is a master regulator of CAR T memory programming [RNA_FOXO1_KO] |
| GSE255416 |
FOXO1 is a master regulator of CAR T memory programming |
|
| Relations |
| BioSample |
SAMN39894060 |
| SRA |
SRX23580502 |
