|
| Status |
Public on Feb 29, 2024 |
| Title |
HA_FOXO1-WT_56 |
| Sample type |
SRA |
| |
|
| Source name |
HA.28z CD4 CAR T cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: HA.28z CD4 CAR T cells
treatment: FOXO1
|
| Treatment protocol |
Cells were treated the same, the only difference being which viruses they were transduced with (CAR/transcription factor)
|
| 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. For experiments requiring magnetic sorting, isolations were performed using Miltenyi MACS or StemCell EasySep magnetic sorting systems. 5e5-1e6 TCs were pelleted and flash frozen prior to RNA seq prep
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated with Trizol, followed by precipitation with isopropanol. RNA was washed with 75% Ethanol and pellet was dried and dissolved in nulcease-free water.
Approximately 20ng total RNA was used for library preparation with Ovation Ultralow RNA-seq V2 (Tecan) from two biological replicates. Libraries were generated according to the manufacturer’s instructions. Approximately 50ng amplified cDNA was subjected to Ovation Ultralow V2 library generation and manufacturer’s instructions were followed. Libraries were size selected with E-Gel EX 2% agarose gels (Life Technologies) and purified by QIAquick Gel Extraction Kit (Qiagen). Libraries were sequenced on HiSeq 6000 instrument.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| 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) |
| GSE255408 |
FOXO1 is a master regulator of CAR T memory programming [RNA_FOXO1_OE_CD4] |
| GSE255416 |
FOXO1 is a master regulator of CAR T memory programming |
|
| Relations |
| BioSample |
SAMN39893849 |
| SRA |
SRX23579549 |
