|
| Status |
Public on May 13, 2021 |
| Title |
01_Donor1_sgControl_NoStim |
| Sample type |
SRA |
| |
|
| Source name |
Primary CD4+ T cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Primary CD4+ T cells
sgrna: No-Target Control
treatment: None
|
| Treatment protocol |
One day after activation, Bulk T cells were transduced with mCherry-2A-dCas9-VP64 lentivirus. Two days after activation, T cells were split into two populations and transduced with either a non-targeting control, or FOXQ1 transcriptional start site targeting sgRNA lentivirus. The following day 2ug/ml puromycin was added to the culture medium and cells were passaged every two days maintaining a minimum concentration of 0.3e6cells/ml. Eight days after initial activation, cells were sorted into CD4+mCherry+ populations and resumed culture in X-VIVO 15 without supplements. The next day cells were treated with 6.25ul/ml anti-CD3/CD28/CD2 Immunocult (Stim) or left untreated (NoStim). Cells were harvest 24 hours after stimulation.
|
| Growth protocol |
Primary human bulk T cells were cultured in X-VIVO 15 (Lonza Bioscience cat 04-418Q) supplemented with 5% FCS, 55mM 2-mercaptoethanol, 4mM N-Acetyl L-Cysteine, and 500 IU/ml recombinant human IL-2 (Amerisource Bergen cat 10101641). Primary T cell activation was done by anti-humanCD3/CD28 CTS dynabeads (Fisher Scientific cat 40203D) at a 1:1 cell to bead ratio at 1e6 cells/ml.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were lysed and RNA was purified using Quick-RNA Microprep kit (Zymo Research) without the optional in-well DNase treatment step. Purified RNA was treated with TURBO DNase (Thermofisher Scientific) to remove potential contaminating DNA. RNA was subsequently purified using RNA Clean & Concentrator-5 kit (Zymo Research). RNA quality control was performed using an RNA ScreenTape assay (Agilent), with all samples having a RNA integrity number > 7.
RNA-seq libraries were prepared using the Illumina Stranded mRNA Prep kit, with 100ng of input RNA, following manufacturer's recommended protocol.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Adapters were trimmed from fastq files using cutadapt version 2.10 with default settings keeping a minimum read length of 20 bp.
Reads were mapped to the human genome GRCh38 keeping only uniquely mapping reads using STAR version 2.7.5b with the following settings “--outFilterMultimapNmax 1”.
Reads overlapping genes were then counted using featureCounts version 2.0.1 with the following settings “-s 2” and using the Gencode version 35 basic transcriptome annotation.
Genome_build: GRCh38
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample
|
| |
|
| Submission date |
May 11, 2021 |
| Last update date |
May 14, 2021 |
| Contact name |
Zachary Steinhart |
| E-mail(s) |
zachary.steinhart@gladstone.ucsf.edu
|
| Organization name |
J. David Gladstone Institutes
|
| Department |
Gladstone-UCSF Institute of Genomic Immunology
|
| Lab |
Marson Lab
|
| Street address |
1650 Owens St
|
| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94158 |
| Country |
USA |
| |
|
| Platform ID |
GPL18573 |
| Series (2) |
| GSE174284 |
CRISPR activation and interference screens decode stimulation responses in primary human T cells [Bulk RNA-seq] |
| GSE174292 |
CRISPR activation and interference screens decode stimulation responses in primary human T cells |
|
| Relations |
| BioSample |
SAMN19110414 |
| SRA |
SRX10863200 |
